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Article

Brain-Derived Neurotrophic Factor Deficiency Exacerbates Innate Immune Responses by Enhancing NLRP3 Inflammasome Activation and GSDMD-Mediated Pyroptosis in Mice

by
Şeniz Erdem
1,2,*,
Neslihan Sağlam
1,
Elif Şahin
1,
Mehmet Erdem
3,
İsmail Abidin
4 and
Ahmet Alver
1
1
Department of Medical Biochemistry, Faculty of Medicine, Karadeniz Technical University, Trabzon 61080, Türkiye
2
Department of Medical Biochemistry, Graduate School of Medical Science, Karadeniz Technical University, Trabzon 61080, Türkiye
3
Department of Medical Biochemistry, Faculty of Medicine, Malatya Turgut Özal University, Malatya 44210, Türkiye
4
Department of Biophysics, Faculty of Medicine, Karadeniz Technical University, Trabzon 61080, Türkiye
*
Author to whom correspondence should be addressed.
Medicina 2026, 62(2), 384; https://doi.org/10.3390/medicina62020384
Submission received: 22 January 2026 / Revised: 4 February 2026 / Accepted: 11 February 2026 / Published: 14 February 2026
(This article belongs to the Section Genetics and Molecular Medicine)
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Abstract

Background and Objectives: The NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome is a key innate immune complex, and its aberrant activation contributes to metabolic and neurodegenerative diseases. Brain-derived neurotrophic factor (BDNF) is a neurotrophin with anti-inflammatory and metabolic regulatory functions, but its role in NLRP3 inflammasome activation and gasdermin D (GSDMD)-mediated pyroptosis remains unclear. The aim of this study was to investigate the effects of BDNF deficiency on LPS- and nigericin-induced NLRP3 inflammasome activation and GSDMD-mediated pyroptosis in vivo, and to elucidate the involvement of NF-κB signaling, autophagy, and ESCRT-III-dependent plasma membrane repair in this process. Materials and Methods: In this in vivo study, male Bdnf +/+ and Bdnf +/− mice were subjected to lipopolysaccharide (LPS) plus nigericin-induced NLRP3 inflammasome activation. Serum and hippocampus, cortex, liver, epididymal adipose, and muscle tissues were collected 24 h after stimulation for analysis of inflammasome-related, autophagy-related, and membrane repair-related proteins by Western blotting and of serum BDNF, interleukin-1β (IL-1β), and interleukin-18 (IL-18) by ELISA. Results: Bdnf +/− mice displayed significantly reduced circulating BDNF levels and exhibited exaggerated LPS plus nigericin-induced increases in IL-1β and IL-18 compared with Bdnf +/+ mice. Across all tissues, BDNF deficiency enhanced NF-κB p65, NLRP3, active caspase-1 p20, and GSDMD expression, indicating amplified inflammasome activation and pyroptosis. Conversely, LC3B and SQSTM1/p62 levels were decreased, and VPS4A expression, a key component of the ESCRT-III membrane repair machinery, was suppressed in Bdnf +/− mice, suggesting impaired selective autophagy, autophagosome formation, and plasma membrane repair. Conclusions: Together, these findings indicate that BDNF restrains NLRP3 inflammasome activation and GSDMD-mediated pyroptosis through inhibition of NF-κB signaling and coordinated activation of autophagy and ESCRT-III-dependent membrane repair. BDNF thus emerges as an endogenous negative regulator of inflammasome activity and a potential therapeutic target for conditions characterized by aberrant NLRP3-driven inflammation.

1. Introduction

The NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome is an intracellular multiple-protein complex activated in response to various physiological/ pathogenic stimuli or tissue damage and plays a central role in innate immunity and inflammation [1,2]. This inflammasome complex is composed of NLRP3, apoptosis-associated speck-like protein (ASC) adapter protein, and pro-caspase-1 [3]. NLRP3 protein acts as a “sensor protein” by detecting cellular alterations induced by pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharide (LPS) and damage-associated molecular patterns (DAMPs), including adenosine triphosphate (ATP) and pore-forming toxins such as nigericin [1,4,5,6,7,8]. Activation of the NLRP3 inflammasome requires two signals. First, the priming signal is induced by pattern recognition receptors (PRRs), such as toll-like receptors (TLRs). TLRs are activated by PAMPs, such as LPS, or other endogenous factors and mechanisms. This signal promotes nuclear factor-kappa B (NF-κB)-mediated transcription of pro-interleukin-1β (pro-IL-1β), pro-interleukin-18 (pro-IL-18), and NLRP3 [4,5,6,7,8]. The activation signal, which is initiated by recognition of an NLRP3 activator such as PAMPs or DAMPs, involves the assembly of the multiprotein complex and fully induces the inflammasome activation [9,10]. The activated NLRP3 inflammasome complex induces self-cleavage of caspase-1 and mediates the cleavage of pro-IL-1β and pro-IL-18 into their mature and biologically active forms, respectively, interleukin-1β (IL-1β) and interleukin-18 (IL-18) [3,11,12]. It also mediates cleavage of gasdermin D (GSDMD). The cleavage process produces an N-terminal fragment that promotes the formation of pores in the plasma membrane, causing loss of membrane integrity and release of the activated IL-1β and IL-18. GSDMD pores cause pyroptosis, which is characterized by plasma membrane rupture, release of mature cytokines, and propagation of the immune response [13].
Activation of NLRP3 inflammasome and GSDMD-mediated pyroptosis can be negatively regulated through different molecular pathways involving selective autophagy activation and endosomal sorting complexes required for transport-III (ESCRT-III)-mediated plasma membrane repair. Autophagy is a necessary mechanism for maintaining cellular homeostasis during stress conditions [14]. Autophagy is regulated by a wide range of proteins, including beclin1, sirtuin 1 (SIRT1), sequestosome 1 (SQSTM1)/p62, and light chain 3 (LC3) A and B. Among these, LC3B and SQSTM1/p62 are commonly utilized to monitor autophagy activity. LC3B plays a role in autophagosome formation, while SQSTM1/p62 acts as a selective autophagy substrate [15]. Autophagy dysfunction can lead to cellular responses such as hyperinflammation and excessive activation of inflammasomes [16,17]. Autophagy can regulate inflammasome activation through multiple mechanisms, including the removal of stimulants that activate inflammasomes from the cytosol, the removal of inflammasomes and their downstream cytokines, and the direct or selective degradation of NLRP3 inflammasome components [17,18,19]. The ESCRT-III machinery repairs damaged plasma membranes caused by GSDMD-N-mediated pyroptosis to maintain membrane integrity. This system comprises ESCRT-III and vacuolar protein sorting 4 (VPS4A, VPS4B) proteins [20]. Following inflammasome activation, GSDMD pores on the plasma membrane are subjected to repair by the ESCRT-III-VPS4A machinery [13]. This process limits both pro-inflammatory cytokine (IL-1β and IL-18) secretion and pyroptotic cell death [21,22].
The NLRP3 inflammasome is essential for innate immunity. However, abnormal NLRP3 inflammasome activation is associated with pathology in several human diseases, such as cancer and metabolic, cardiovascular, neurodegenerative, and inflammatory diseases [1,23,24,25]. Therefore, NLRP3 inflammasome activation must be strictly controlled to maintain immune system homeostasis and avoid deleterious consequences [26]. Despite these extensive studies on NLRP3 inflammasome involvement in human health and disease, its endogenous regulatory networks remain largely unknown. Therefore, gaining insight into the mechanisms that negatively regulate inflammasome activation is crucial for developing novel therapeutic approaches for these diseases [27].
Brain-derived neurotrophic factor (BDNF) is among the most extensively studied neuronal growth factors and a crucial neurotrophin that plays a vital role in the development, plasticity, and survival of neurons in the central and peripheral nervous system [25,28,29,30]. It is also involved in various regulatory processes, including energy homeostasis, the brain, and glucose metabolism [30,31,32,33]. BDNF is widely expressed in the brain and peripheral tissues (especially the liver, skeletal muscle, and adipose tissue). Therefore, these tissues were chosen for this study because BDNF is both highly expressed in these tissues and is involved in processes such as the regulation of energy metabolism. Its dysregulation is implicated in the pathophysiology of a wide range of neurodegenerative and metabolic disorders [34,35,36,37]. In addition, BDNF also regulates inflammatory homeostasis, inhibiting NF-κB activation and modulating the production and release of pro-inflammatory cytokines, including tumor necrosis factor (TNF), interleukin-6 (IL-6), interferon-γ, and IL-1β [38,39,40,41,42]. However, despite the increasing number of studies focusing on the effects of BDNF in immunomodulatory activity, there are a very limited number of studies investigating the effect of BDNF on NLRP3 inflammasome activation and GSDMD-mediated pyroptosis.
In this investigation, the effects of BDNF on LPS and nigericin-induced NLRP3 inflammasome activation, GSDMD-mediated pyroptotic cell death, and negative regulators of this pathway were investigated in vivo in Bdnf +/− mice.

2. Materials and Methods

2.1. Animals, Housing Conditions, and Mice Genotyping

A total of n = 30 male Bdnf +/+ and n = 30 Bdnf +/− mice, aged 8–10 weeks, were provided by Karadeniz Technical University, Surgical Application and Research Center. Mouse strains were maintained and housed under controlled conditions at 20–24 °C and relative humidity on a 12:12 h light/dark cycle, with food and water provided ad libitum. Genomic DNA was extracted from mouse-tail biopsy samples for genotyping. Mouse genotyping was performed using a standard polymerase chain reaction (PCR) [43]. The following primers were used: Bdnf forward primer (5′-ACCATAAGGACGCGGACTTGTAC-3′), neomycin forward primer (5′-GATTCGCAGCGCATCGCCTT-3′), and reverse primer (5′-GAAGTGTCTATCCTTATGAATCGC-3′). Bdnf +/− mice possess only one functional allele for the gene, as Bdnf +/− mice lack one of the two Bdnf coding alleles. The gene encoding Bdnf is replaced by a neomycin gene. Bdnf +/− mice were capable of reproduction and did not exhibit morphological abnormalities. Bdnf −/− mutant mice die within 2 days after birth due to large-scale losses of sensory neurons in the petrosal–nodose ganglia. For this reason, Bdnf +/− mice were used instead of Bdnf −/− mice. All experimental procedures were conducted in accordance with institutional and national guidelines for the care and use of laboratory animals. Animal discomfort and stress were minimized throughout the study. Tail biopsy for genotyping was performed using standard, minimally invasive procedures. No surgical interventions were applied; therefore, postoperative analgesia was not required. Animals were monitored daily for signs of pain or distress, and no unexpected adverse events were observed.
In the Section 2, all procedures applied to the mice and the experiments performed are summarized in Figure 1.

2.2. In Vivo NLRP3 Inflammasome Activation

Mice were randomly divided into six groups with 10 animals per group: Group 1 (Bdnf +/+) animals were injected with physiological saline solution intraperitoneally; Group 2 (Bdnf +/+ + Ethanol) animals were injected with 5% (v/v) ethanol (as solvent of nigericin) intraperitoneally; Group 3 (Bdnf +/+ + LPS + Nigericin) animals were injected intraperitoneally with 5 mg/kg LPS from Escherichia coli O55:B5 (Sigma-Aldrich, Saint Louis, MO, USA), and then after 3 h were injected intraperitoneally with 1 mg/kg Nigericin (Cayman Chemical, Ann Arbor, MI, USA); Group 4 (Bdnf +/−) animals were injected with physiological saline solution intraperitoneally; Group 5 (Bdnf +/− + Ethanol) animals were injected with ethanol intraperitoneally; and Group 6 (Bdnf +/− + LPS + Nigericin) animals were injected intraperitoneally with 5 mg/kg LPS, and then after 3 h were injected intraperitoneally with 1 mg/kg Nigericin. After 24 h, all mice were sacrificed by decapitation; blood samples were collected into a serum collection tube and stored until analysis. The hippocampus, cortex, liver, epididymal adipose, and muscle tissues were quickly separated, flash-frozen in liquid nitrogen, and stored at −80 °C in a deep freezer for biochemical analysis.

2.3. Western Blotting Analysis

Proteins were extracted from the hippocampus, cortex, liver, epididymal adipose, and muscle tissues of mice in each group, as mentioned above, and processed for Western blotting analysis. Total proteins from frozen tissues were isolated with the cold RIPA lysis buffer (Serva, Heidelberg, Germany), including protease and phosphatase inhibitor cocktail (Serva, Germany) for 1 h on ice and then centrifuged at 20,000× g for 25 min at 4 °C. Protein concentrations were detected by the BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA). The extracted proteins were stored at −80 °C in a deep freezer. An equal amount of proteins (25–30 μg of protein of each sample) was denatured at 99 °C for 5 min and separated using 10–12% SDS-PAGE. Then, the proteins were transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). The nitrocellulose membranes used for Western blotting had a pore size of 0.45 µm. The membranes were blocked in 5% non-fat dry milk in Tris-buffered saline containing 0.05% Tween-20 (TBS-T) for 1 h at room temperature and then were incubated at 4 °C for overnight with the primary antibodies: anti-NF-κB p65 (8242S, Cell Signaling Technology, Danvers, MA, USA), anti-NLRP3 (ab263899, Abcam, Cambridge, Cambridgeshire, UK), anti-caspase-1 p20 (AG-20B-0042, AdipoGen, Füllinsdorf, Switzerland), anti-GSDMD (ab219800, Abcam, Cambridge, Cambridgeshire, UK), anti-LC3B (83506S, Cell Signaling Technology, Danvers, MA, USA), anti-SQSTM1/p62 (23214S, Cell Signaling Technology, Danvers, MA, USA), anti-VPS4A (sc-393428, Santa Cruz, Dallas TX, USA), anti-β-actin (3700S, Cell Signaling Technology, Danvers, MA, USA), and anti-GAPDH (2118S, Cell Signaling Technology, Danvers, MA, USA), all used at a dilution of 1:1000. The next day, the membranes were washed three times with TBS-T and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (goat anti-rabbit HRP (170-5046, Bio-Rad, Hercules, CA, USA) or goat anti-mouse HRP (170-5047, Bio-Rad, Hercules, CA, USA)) at a dilution of 1:5000 at room temperature for 1 h. Finally, the protein signals were screened with ClarityTM Western ECL Substrate (170-5060, Bio-Rad, Hercules, CA, USA) using a densitometer (Bio-Rad Chemidoc MP Imaging System, Hercules, CA, USA). Band density analysis was performed with Image Lab™ software version 6.0.1 (Bio-Rad Laboratories, Hercules, CA, USA). Results were normalized to β-actin or GAPDH for quantification.

2.4. Enzyme-Linked Immunosorbent Assay (ELISA)

Brain-derived neurotrophic factor and pro-inflammatory cytokine (IL-1β and IL-18) levels in the serum were measured using mouse BDNF (Abnova, Taipei, Taiwan), IL-1β, and IL-18 sandwich ELISA kits (Invitrogen, Vienna, Austria), following the manufacturer’s protocol. The absorbance was measured at 450 nm in a microplate spectrophotometer (Molecular Devices VersaMax, San Jose, CA, USA).

2.5. Statistical Analysis

The analysis of the experimental data was performed on the SPSS 25 (IBM Statistics 25, New York, NY, USA) program. BDNF, IL-1β, and IL-18 data were given in [median (IQR)] quartiles of 25–75%. The graphics were conveyed using boxplots. Other data were expressed as mean ± standard error of the mean (mean ± S.E.M.). Normality analysis of the data was assessed by the Kolmogorov–Smirnov test. Statistical difference between groups was determined by Kruskal–Wallis analysis of variance. Mann–Whitney U was used for comparisons between two normally distributed groups. p < 0.05 values were considered statistically significant.

3. Results

3.1. Circulating BDNF Concentrations

Serum BDNF levels were determined to demonstrate the absence of BDNF in Bdnf +/− mice, and these were compared with those of Bdnf +/+ mice. BDNF levels were significantly lower in Bdnf +/− mice compared to Bdnf +/+ mice (p < 0.001). Serum BDNF levels were significantly decreased in LPS plus nigericin-injected Bdnf +/+ and Bdnf +/− mice (p = 0.001 and p = 0.004), and serum BDNF levels in LPS plus nigericin-injected Bdnf +/− mice were significantly lower compared with LPS plus nigericin-injected Bdnf +/+ mice (p = 0.01) (Figure 2A).

3.2. Bdnf Deficiency Increases NLRP3 Inflammasome-Related Pro-Inflammatory Cytokines Il-1β and Il-18 In Vivo

As a result of NLRP3 inflammasome activation, IL-1β and IL-18 cytokines are produced, which are pro-inflammatory molecules. To test whether BDNF could block NLRP3 activation, we examined the impact of BDNF on IL-1β and IL-18 secretion. We found that IL-1β levels in Bdnf +/− mice were significantly increased compared with Bdnf +/+ mice (p = 0.016). Results indicated that IL-1β levels were significantly elevated in LPS plus nigericin-injected Bdnf +/+ and Bdnf +/− mice (p < 0.001 and p < 0.001), and this reversed in Bdnf +/+ mice (p = 0.002) (Figure 2B). Lastly, IL-18 levels in Bdnf +/− mice were increased significantly compared with Bdnf +/+ mice (p = 0.016). Results indicated that IL-18 levels were statistically significantly elevated in LPS plus nigericin-injected Bdnf +/+ and Bdnf +/− mice (p < 0.001 and p < 0.001), and this reversed in Bdnf +/+ mice (p = 0.002) (Figure 2C).

3.3. Bdnf Deficiency Promotes NLRP3 Inflammasome Activation and GSDMD-Mediated Pyroptosis Through Enhancing NF-κB Activation in Hippocampus, Cortex, Liver, Epididymal Adipose, and Muscle Tissues

Nuclear factor-kappa B pathway, which is involved in the priming signal in NLRP3 inflammasome activation, stimulates transcription of NLRP3, pro-IL-1β, and pro-IL-18. The formation of the inflammasome molecule, which consists of NLRP3, ASC, and pro-caspase-1 proteins, ultimately results in autocatalysis and activation of caspase-1. Enzymatically activated caspase-1 converts pro-IL-1β and pro-IL-18 cytokines to their mature forms (IL-1β and IL-18). Active caspase-1 also mediates the proteolytic cleavage of GSDMD, resulting in the formation of GSDMD-N. GSDMD-N forms membrane pores that lead to pyroptosis. Pyroptotic cells contribute to the spread of inflammation by secreting molecules such as IL-1β and IL-18. For this purpose, NLRP3, active caspase-1 p20, GSDMD, and NF-κB p65 protein levels were investigated in hippocampus, cortex, liver, epididymal adipose, and muscle tissues (Figure 3, Figure 4, Figure 5, Figure 6 and Figure 7). In hippocampus tissues, as shown by Western blotting, NLRP3, active caspase-1 p20, GSDMD, and NF-κB p65 expression levels in Bdnf +/− mice were significantly increased compared with Bdnf +/+ mice (respectively, p = 0.007, p = 0.016, p = 0.006, and p = 0.037). In LPS plus nigericin-injected Bdnf +/+ mice, we determined that NLRP3, active caspase-1 p20, GSDMD, and NF-κB p65 expression levels were significantly elevated compared with Bdnf +/+ mice (respectively, p = 0.002, p = 0.004, p = 0.004, and p = 0.004). In LPS plus nigericin-injected Bdnf +/− mice, we determined that NLRP3, active caspase-1 p20, GSDMD, and NF-κB p65 expression levels were significantly elevated compared with Bdnf +/− mice (respectively, p = 0.004, p = 0.004, p = 0.004, and p = 0.004). This rise was reversed in Bdnf +/+ mice (respectively, p = 0.004, p = 0.016, p = 0.004, and p = 0.004) (Figure 3A–E).
In cortex tissues, NLRP3, active caspase-1 p20, GSDMD, and NF-κB p65 expression levels in Bdnf +/− mice were significantly increased compared with Bdnf +/+ mice (respectively, p = 0.004, p = 0.01, p = 0.025, and p = 0.016). In LPS plus nigericin-injected Bdnf +/+ mice, we determined that NLRP3, active caspase-1 p20, GSDMD, and NF-κB p65 expression levels were significantly elevated compared with Bdnf +/+ mice (respectively, p = 0.004, p = 0.004, p = 0.004, and p = 0.004). In LPS plus nigericin-injected Bdnf +/− mice, we determined that NLRP3, active caspase-1 p20, GSDMD, and NF-κB p65 expression levels were significantly elevated compared with Bdnf +/− mice (respectively, p = 0.004, p = 0.004, p = 0.004, and p = 0.006). This rise was reversed in Bdnf +/+ mice (respectively, p = 0.004, p = 0.004, p = 0.004, and p = 0.037) (Figure 4A–E).
In liver tissues, NLRP3, active caspase-1 p20, GSDMD, and NF-κB p65 expression levels in Bdnf +/− mice were significantly increased compared with Bdnf +/+ mice (respectively, p = 0.004, p = 0.004, p = 0.004, and p = 0.037). In LPS plus nigericin-injected Bdnf +/+ mice, we determined that NLRP3, active caspase-1 p20, GSDMD, and NF-κB p65 expression levels were significantly elevated compared with Bdnf +/+ mice (respectively, p = 0.004, p = 0.004, p = 0.004, and p = 0.004). In LPS plus nigericin-injected Bdnf +/− mice, we determined that NLRP3, active caspase-1 p20, GSDMD, and NF-κB p65 expression levels were significantly elevated compared with Bdnf +/− mice (respectively, p = 0.004, p = 0.004, p = 0.004, and p = 0.006). This rise was reversed in Bdnf +/+ mice (respectively, p = 0.004, p = 0.004, p = 0.01, and p = 0.025) (Figure 5A–E).
In epididymal adipose tissues, NLRP3, active caspase-1 p20, GSDMD, and NF-κB p65 expression levels in Bdnf +/− mice were significantly increased compared with Bdnf +/+ mice (respectively, p = 0.004, p = 0.025, p = 0.037, and p = 0.004). In LPS plus nigericin-injected Bdnf +/+ mice, we determined that NLRP3, active caspase-1 p20, GSDMD, and NF-κB p65 expression levels were significantly elevated compared with Bdnf +/+ mice (respectively, p = 0.004, p = 0.004, p = 0.004, and p = 0.004). In LPS plus nigericin-injected Bdnf +/− mice, we determined that NLRP3, active caspase-1 p20, GSDMD, and NF-κB p65 expression levels were significantly elevated compared with Bdnf +/− mice (respectively, p = 0.004, p = 0.004, p = 0.004, and p = 0.004). This rise was reversed in Bdnf +/+ mice (respectively, p = 0.016, p = 0.004, p = 0.004, and p = 0.004) (Figure 6A–E).
In muscle tissues, NLRP3, active caspase-1 p20, GSDMD, and NF-κB p65 expression levels in Bdnf +/− mice were significantly increased compared with Bdnf +/+ mice (respectively, p = 0.016, p = 0.006, p = 0.025, and p = 0.006). In LPS plus nigericin-injected Bdnf +/+ mice, we determined that NLRP3, active caspase-1 p20, GSDMD, and NF-κB p65 expression levels were significantly elevated compared with Bdnf +/+ mice (respectively, p = 0.004, p = 0.004, p = 0.004, and p = 0.004). In LPS plus nigericin-injected Bdnf +/− mice, we determined that NLRP3, active caspase-1 p20, GSDMD, and NF-κB p65 expression levels were significantly elevated compared with Bdnf +/− mice (respectively, p = 0.004, p = 0.004, p = 0.004, and p = 0.004). This rise was reversed in Bdnf +/+ mice (respectively, p = 0.01, p = 0.006, p = 0.025, and p = 0.016) (Figure 7A–E).

3.4. Bdnf Promotes SQSTM1/p62-Dependent Selective Autophagy and LC3B-Mediated Autophagosome Formation to Inhibit NLRP3 Inflammasome Activation in Hippocampus, Cortex, Liver, Epididymal Adipose, and Muscle Tissues

Autophagy may suppress cell pyroptosis and the release of inflammatory cytokines by degrading inflammasomes and other crucial components involved in pyroptosis. Autophagy can reduce excessive NLRP3 inflammasome activation. LC3B and SQSTM1/p62 are prevalently used autophagy markers for the autophagy process. In an in vivo model, we examined whether BDNF would negatively regulate NLRP3 inflammasome activation by enhancing autophagy in response to LPS plus nigericin. To confirm this, LC3B and SQSTM1/p62 protein levels were investigated in hippocampus, cortex, liver, epididymal adipose, and muscle tissues (Figure 8). In hippocampus tissues, as shown by Western blotting, LC3B and SQSTM1/p62 expression levels in Bdnf +/− mice were significantly decreased compared with Bdnf +/+ mice (respectively, p = 0.004 and p = 0.004). In LPS plus nigericin-injected Bdnf +/+ mice, we determined that LC3B and SQSTM1/p62 expression levels were significantly increased compared with Bdnf +/+ mice (respectively, p = 0.025 and p = 0.013). In LPS plus nigericin-injected Bdnf +/− mice, we determined that LC3B and SQSTM1/p62 expression levels were significantly increased compared with Bdnf +/− mice (respectively, p = 0.037 and p = 0.004). LPS plus nigericin-injected Bdnf +/+ mice had improved autophagy markers compared with LPS plus nigericin-injected Bdnf +/− mice (respectively, p = 0.004 and p = 0.004) (Figure 8A–C). In cortex tissues, LC3B and SQSTM1/p62 expression levels in Bdnf +/− mice were significantly decreased compared with Bdnf +/+ mice (respectively, p = 0.01 and p = 0.004). In LPS plus nigericin-injected Bdnf +/+ mice, we determined that LC3B and SQSTM1/p62 expression levels were significantly increased compared with Bdnf +/+ mice (respectively, p = 0.025 and p = 0.004). In LPS plus nigericin-injected Bdnf +/− mice, we determined that LC3B and SQSTM1/p62 expression levels were significantly increased compared with Bdnf +/− mice (respectively, p = 0.025 and p = 0.025). LPS plus nigericin-injected Bdnf +/+ mice had improved autophagy markers compared with LPS plus nigericin-injected Bdnf +/− mice (respectively, p = 0.004 and p = 0.004) (Figure 8D–F). In liver tissues, LC3B and SQSTM1/p62 expression levels in Bdnf +/− mice were significantly decreased compared with Bdnf +/+ mice (respectively, p = 0.004 and p = 0.016). In LPS plus nigericin-injected Bdnf +/+ mice, we determined that LC3B and SQSTM1/p62 expression levels were significantly increased compared with Bdnf +/+ mice (respectively, p = 0.004 and p = 0.01). In LPS plus nigericin-injected Bdnf +/− mice, we determined that LC3B and SQSTM1/p62 expression levels were significantly increased compared with Bdnf +/− mice (respectively, p = 0.025 and p = 0.016). LPS plus nigericin-injected Bdnf +/+ mice had improved autophagy markers compared with LPS plus nigericin-injected Bdnf +/− mice (respectively, p = 0.004 and p = 0.004) (Figure 8G–I). In epididymal adipose tissues, LC3B and SQSTM1/p62 expression levels in Bdnf +/− mice were significantly decreased compared with Bdnf +/+ mice (respectively, p = 0.01 and p = 0.004). In LPS plus nigericin-injected Bdnf +/+ mice, we determined that LC3B and SQSTM1/p62 expression levels were significantly increased compared with Bdnf +/+ mice (respectively, p = 0.01 and p = 0.025). In LPS plus nigericin-injected Bdnf +/− mice, we determined that LC3B and SQSTM1/p62 expression levels were significantly increased compared with Bdnf +/− mice (respectively, p = 0.01 and p = 0.016). LPS plus nigericin-injected Bdnf +/+ mice had improved autophagy markers compared with LPS plus nigericin-injected Bdnf +/− mice (respectively, p = 0.006 and p = 0.004) (Figure 8J–L). In muscle tissues, LC3B and SQSTM1/p62 expression levels in Bdnf +/− mice were significantly decreased compared with Bdnf +/+ mice (respectively, p = 0.004 and p = 0.006). In LPS plus nigericin-injected Bdnf +/+ mice, we determined that LC3B and SQSTM1/p62 expression levels were significantly increased compared with Bdnf +/+ mice (respectively, p = 0.006 and p = 0.016). In LPS plus nigericin-injected Bdnf +/− mice, we determined that LC3B and SQSTM1/p62 expression levels were significantly increased compared with Bdnf +/− mice (respectively, p = 0.004 and p = 0.037). LPS plus nigericin-injected Bdnf +/+ mice had improved autophagy markers compared with LPS plus nigericin-injected Bdnf +/− mice (respectively, p = 0.004 and p = 0.004) (Figure 8M–O).

3.5. Bdnf Activated the ESCRT-III-Mediated Plasma Membrane Repair System to Prevent Pyroptosis in Hippocampus, Cortex, Liver, Epididymal Adipose, and Muscle Tissues

In the pyroptotic cells, the ESCRT-III-VPS4A system removes GSDMD pores from the plasma membrane and limits pyroptotic cell death. Thus, we next looked at whether BDNF would negatively regulate NLRP3 inflammasome activation via the activated ESCRT-III-mediated plasma membrane repair system in response to LPS in combination with nigericin. To verify this, VPS4A protein levels were investigated in hippocampus, cortex, liver, epididymal adipose, and muscle tissues (Figure 9). In hippocampus tissues, as shown by Western blotting, VPS4A expression levels in Bdnf +/− mice were significantly decreased compared with Bdnf +/+ mice (p = 0.004). In LPS plus nigericin-injected Bdnf +/+ mice, we determined that VPS4A expression levels were significantly decreased compared with Bdnf +/+ mice (p = 0.025). In LPS plus nigericin-injected Bdnf +/− mice, we determined that VPS4A expression levels were significantly decreased compared with Bdnf +/− mice (p = 0.025). LPS plus nigericin-injected Bdnf +/+ mice improved VPS4A protein levels compared with LPS plus nigericin-injected Bdnf +/− mice (p = 0.025) (Figure 9A,B). In cortex tissues, VPS4A expression levels in Bdnf +/− mice were significantly decreased compared with Bdnf +/+ mice (p = 0.004). In LPS plus nigericin-injected Bdnf +/+ mice, we determined that VPS4A expression levels were significantly decreased compared with Bdnf +/+ mice (p = 0.025). In LPS plus nigericin-injected Bdnf +/− mice, we determined that VPS4A expression levels were significantly decreased compared with Bdnf +/− mice (p = 0.004). LPS plus nigericin-injected Bdnf +/+ mice had improved VPS4A protein levels compared with LPS plus nigericin-injected Bdnf +/− mice (p = 0.004) (Figure 9C,D). In liver tissues, VPS4A expression levels in Bdnf +/− mice were significantly decreased compared with Bdnf +/+ mice (p = 0.004). In LPS plus nigericin-injected Bdnf +/+ mice, we determined that VPS4A expression levels were significantly decreased compared with Bdnf +/+ mice (p = 0.016). In LPS plus nigericin-injected Bdnf +/− mice, we determined that VPS4A expression levels were significantly decreased compared with Bdnf +/− mice (p = 0.004). LPS plus nigericin-injected Bdnf +/+ mice had improved VPS4A protein levels compared with LPS plus nigericin-injected Bdnf +/− mice (p = 0.004) (Figure 9E,F). In epididymal adipose tissues, VPS4A expression levels in Bdnf +/− mice were significantly decreased compared with Bdnf +/+ mice (p = 0.004). In LPS plus nigericin-injected Bdnf +/+ mice, we determined that VPS4A expression levels were significantly decreased compared with Bdnf +/+ mice (p = 0.006). In LPS plus nigericin-injected Bdnf +/− mice, we determined that VPS4A expression levels were significantly decreased compared with Bdnf +/− mice (p = 0.037). LPS plus nigericin-injected Bdnf +/+ mice had improved VPS4A protein levels compared with LPS plus nigericin-injected Bdnf +/− mice (p = 0.004) (Figure 9G,H). In muscle tissues, VPS4A expression levels in Bdnf +/− mice were significantly decreased compared with Bdnf +/+ mice (p = 0.004). In LPS plus nigericin-injected Bdnf +/+ mice, we determined that VPS4A expression levels were significantly decreased compared with Bdnf +/+ mice (p = 0.01). In LPS plus nigericin-injected Bdnf +/− mice, we determined that VPS4A expression levels were significantly decreased compared with Bdnf +/− mice (p = 0.006). LPS plus nigericin-injected Bdnf +/+ mice had improved VPS4A protein levels compared with LPS plus nigericin-injected Bdnf +/− mice (p = 0.004) (Figure 9I,J).

4. Discussion

The study we conducted on Bdnf +/− mice demonstrated that BDNF attenuated LPS plus nigericin-induced NLRP3 inflammasome activation by inhibiting NF-κB and, consequently, IL-1β and IL-18 production. Additionally, BDNF activated selective autophagy, autophagosome formation, and ESCRT-III-mediated plasma membrane repair pathways, which inhibited NLRP3 inflammasome activation and pyroptosis. Our research has pointed out that BDNF may be a critical endogenous negative regulator of NLRP3 inflammasome activation.
As previously described, the canonical NLRP3 inflammasome activation occurs via two-step signaling: priming and activation signal [44]. NLRP3 inflammasome activation was induced using the LPS and nigericin model in Bdnf +/+ and Bdnf +/− mice. In this case, LPS, commonly used to induce innate immune responses, is a lipophilic molecule and may cross the blood–brain barrier (BBB) [45]. LPS, a well-known PAMP, also acts as the priming signal necessary for classical NLRP3 inflammasome activation via PRR, such as Toll-like receptor 4 (TLR 4) [46]. Nigericin (a K+/H+ ionophore) acts as the activation signal, which induces the NLRP3 inflammasome complex by mediating potassium efflux through pannexin-1 [47,48,49]. In addition, during the activation phase, a variety of molecular factors and cellular signals, including mitochondrial dysfunction, reactive oxygen species production, decreased intracellular potassium levels, disruption of calcium (Ca2+) homeostasis, lysosomal destabilization, Golgi fragmentation, and metabolic alteration pathways, also contribute to NLRP3 inflammasome activation [48]. In our study, molecular tests carried out in Bdnf +/+ and Bdnf +/− mice given LPS plus nigericin injections revealed that NLRP3 inflammasome activation occurred. The accuracy of NLRP3 inflammasome activation was demonstrated by an increase in IL-1β and IL-18 cytokine levels, as well as an increase in NLRP3 inflammasome protein levels in the hippocampus, cortex, liver, epididymal adipose, and muscle tissues (Figure 2, Figure 3, Figure 4, Figure 5, Figure 6 and Figure 7).
NLRP3 inflammasome activation must be tightly regulated to prevent excessive tissue damage and disruption of homeostasis [50]. Several negative regulators, such as leucine-rich repeat Fli-I-interacting protein 2, A20, small heterodimer partner, aryl hydrocarbon receptor, lipin-2, and RKIP, are known to attenuate the activation of inflammasomes through different mechanisms [27,51,52,53,54,55]. These endogenous regulators play a critical role in negatively regulating NLRP3 inflammasome activation, thereby providing crucial protection against excessive inflammation and tissue damage. In this study, we identified BDNF as a negative regulator of the NLRP3 inflammasome in vivo. Here, we showed that BDNF deficiency increases the sensitivity of mice to NLRP3 inflammasome activation. Our results provide data that BDNF is directly associated with the inhibition of NLRP3 inflammasome activation.
Brain-derived neurotrophic factor is a multi-functional neurotrophin that plays crucial roles in neurological activities [56]. However, its dysregulation is also linked to the pathogenesis of inflammatory disorders [34]. Additionally, BDNF mutations and genetic variants (especially BDNF Val66Met polymorphism) are associated with increased inflammation [57,58,59]. Promising strong immunomodulatory functions and anti-inflammatory effects of BDNF have been identified in various diseases or disease models [60]. Likely common factors are the regulation of cytokines, increasing the expression and secretion of anti-inflammatory molecules, while inhibiting pro-inflammatory cytokines, and inhibition of myeloid differentiation primary response gene 88 (MyD88)/NF-κB-signaling pathway or MAPK. BDNF suppresses the production of inflammasome-mediated cytokines, such as IL-1β and IL-18, and inflammasome-independent cytokines, such as TNF-α, IL-6, and interferon-gamma (INF-γ) [39,41,42,61,62]. It also stimulates the production of anti-inflammatory cytokines such as IL-10 and IL-4 [38,41,42]. Parrot et al. have demonstrated that LPS, which triggers an acute pro-inflammatory response, induces exaggerated central pro-inflammatory cytokines in Bdnf +/− mice. In this study, pro-inflammatory cytokines IL-1β, TNF-α, and IL-6 were found to be overexpressed in Bdnf +/− mice relative to Bdnf +/+ littermate control mice [63]. In another study, it was reported that BDNF/tropomyosin receptor kinase B (TrkB) deficiency increased inflammatory cytokines in brain tissues [64]. Consistent with these findings, our results demonstrate that BDNF-deficient mice displayed increased release of LPS plus nigericin-mediated IL-1β and IL-18. BDNF significantly reduced the levels of IL-1β and IL-18, which increase with LPS plus nigericin-mediated damage (p < 0.05). These results indicated that BDNF exhibited inhibitory effects on IL-1β and IL-18 secretion in response to the activation of the NLRP3 inflammasome. Additionally, NF-κB is the crucial transcription factor upregulating pro-IL-1β, pro-IL-18, NLRP3 protein synthesis, and NLRP3 inflammasome activation [7]. Considering the studies showing that BDNF inhibits NF-κB, our results showed that BDNF deficiency significantly increased NF-κB p65 protein levels in the hippocampus, cortex, liver, epididymal adipose, and muscle tissues of the groups, independent of NLRP3 inflammasome activation (p < 0.05). Additionally, it was found that NF-κB p65 protein levels increased significantly with BDNF deficiency in the hippocampus, cortex, liver, epididymal adipose, and muscle tissues of the NLRP3 inflammasome-activated groups (p < 0.05). Consequently, BDNF impairs NLRP3 inflammasome activation via the inhibition of NF-κB (Figure 2, Figure 3, Figure 4, Figure 5, Figure 6 and Figure 7).
The NLRP3 inflammasome activation initiates the process of pyroptosis and promotes the spread of inflammation [65,66]. There has been little research on the impact of BDNF on the control of NLRP3 inflammasome activation. It has been previously shown that BDNF alleviates oxidized LDL-mediated NLRP3 inflammasome activation and NLRP3 inflammasome-mediated pyroptosis in vascular endothelial cells through regulation of KLF2/HK1-dependent glucose metabolism and protection of mitochondrial homeostasis [67]. Previous research has shown that intranasal BDNF treatment reduced inflammation-associated protein levels (NLRP3 and IL-1β) in the hippocampus of chronic unpredictable mild stress (CUMS)-induced depression mice [68]. We investigated the effects of BDNF on the NLRP3 inflammasome activation and GSDMD-mediated pyroptosis in vivo. Correspondingly, our data demonstrate that BDNF deficiency significantly increased NLRP3, caspase-1 p20, and GSDMD protein levels in the hippocampus, cortex, liver, epididymal adipose, and muscle tissues of the groups, independent of NLRP3 inflammasome activation (p < 0.05). Additionally, it was found that NLRP3, caspase-1 p20, and GSDMD protein levels increased significantly with BDNF deficiency in the hippocampus, cortex, liver, epididymal adipose, and muscle tissues of the NLRP3 inflammasome-activated groups (p < 0.05). Consequently, BDNF promotes NLRP3 inflammasome-related activity and GSDMD-mediated pyroptosis (Figure 2, Figure 3, Figure 4, Figure 5, Figure 6 and Figure 7).
Autophagy is a homeostatic, proteolytic process that removes misfolded or damaged proteins and organelles and regulates inflammasome function [69]. Autophagy diminishes hyperinflammation and hyperactivation of inflammasomes. Similarly, inflammasome activation can upregulate autophagy to protect the host from excessive inflammation [70]. Regulation of inflammasome activation by autophagy can occur in multiple ways, through either the removal of endogenous inflammasome activators or the removal of inflammasome components and their downstream cytokines directly. Thus, autophagy acts as a major regulator of inflammasomes [16]. Autophagy has recently been linked to BDNF [14,71]. Activation of the BDNF/TrkB signaling pathway causes the prevention of cell apoptosis, activation of cell autophagy, autophagosome formation (increase in LC3 and p62 levels), and regulation of inflammatory responses under normal physiological conditions [72,73,74]. Our data demonstrate that BDNF deficiency significantly decreased LC3B and SQSTM1/p62 protein levels in the hippocampus, cortex, liver, epididymal adipose, and muscle tissues of the groups, independent of NLRP3 inflammasome activation (p < 0.05). In addition, it was found that LC3B and SQSTM1/p62 protein levels decreased significantly with BDNF deficiency in the hippocampus, cortex, liver, epididymal adipose, and muscle tissues of the NLRP3 inflammasome-activated groups (p < 0.05). According to these results, BDNF deficiency leads to the impairment of autophagy. Additionally, these findings revealed that BDNF alleviated NLRP3 inflammasome activation via enhancing autophagy (Figure 8).
The ESCRT machinery is composed of several functional subunits, including ESCRT-0, -I, -II, and -III, apoptosis-linked gene-2 (ALG-2) interacting protein X (ALIX), and vacuolar protein sorting 4 homolog A (VPS4A) [75]. ESCRT-dependent membrane repair negatively regulates pyroptosis downstream of GSDMD activation [21]. The ESCRT-III-VPS4A system blocks GSDMD-mediated pyroptotic membrane pore formation and negatively regulates IL-1β and IL-18 secretion after NLRP3 inflammasome activation. This process limits pyroptotic cell death [21,22]. Our data demonstrate that BDNF deficiency significantly decreased VPS4A protein levels in the hippocampus, cortex, liver, epididymal adipose, and muscle tissues of the groups, independent of NLRP3 inflammasome activation (p < 0.05). Additionally, it was found that VPS4A protein levels decreased significantly with BDNF deficiency in the hippocampus, cortex, liver, epididymal adipose, and muscle tissues of the NLRP3 inflammasome-activated groups (p < 0.05). Overall, BDNF insufficiency cannot tolerate plasma membrane damage. Additionally, these data further confirm BDNF’s role in suppressing NLRP3 inflammasome activation by activating ESCRT-III-mediated plasma membrane repair (Figure 9).
Although our study demonstrates the involvement of BDNF in promoting autophagy and ESCRT-mediated plasma membrane repair, the precise signaling mechanisms remain to be fully elucidated. BDNF primarily acts through its high-affinity receptor TrkB, activating downstream signaling cascades such as the PI3K/Akt/mTOR and AMPK pathways, both of which play critical roles in the regulation of autophagy flux and mitochondrial homeostasis [71,76]. These pathways influence the initiation of autophagosome formation, as well as the expression of autophagy-related proteins, including LC3B and SQSTM1/p62. Furthermore, BDNF may impact ESCRT-III-mediated plasma membrane repair indirectly through its effects on cytoskeletal remodeling, membrane trafficking, or endosomal dynamics, although this relationship is not yet fully established. Recent evidence suggests that neuronal activity and neurotrophic factors like BDNF can influence membrane remodeling via small GTPases and intracellular trafficking components [77,78]. Further research is required to determine whether BDNF directly regulates components of the ESCRT-III complex, such as VPS4A or CHMP proteins, in the context of inflammation or pyroptosis.
Our findings revealed that BDNF deficiency can increase the immune response independently of inflammasome activation. Also, with NLRP3 inflammasome activation, Bdnf +/− mice were shown to be more sensitive to increased NLRP3 inflammasome activation. Based on the data obtained in our study, it was revealed that BDNF inhibited NLRP3 inflammasome activation and prevented pyroptotic cell death by regulating the NF-κB axis in the LPS plus nigericin-induced NLRP3 inflammasome activation model in Bdnf +/+ and Bdnf +/− mice. We also showed that BDNF has an important role in the activation of selective autophagy, autophagosome formation, and ESCRT-III-mediated plasma membrane repair, which are negative regulators of NLRP3 inflammasome activation and GSDMD-mediated pyroptosis. To our knowledge, this is the first in vivo study to demonstrate that BDNF deficiency amplifies NLRP3 inflammasome activation through coordinated suppression of autophagy and ESCRT machinery. Our results suggest that BDNF may act as a systemic immunomodulatory effect beyond the CNS. Although our results provide strong evidence of BDNF’s regulatory role, future studies using tissue-specific knockouts or pharmacological inhibitors could further clarify the downstream signaling pathways involved. Understanding how BDNF interacts with inflammasome modulators could open new avenues for therapeutic intervention in inflammatory and neurodegenerative diseases.
While this study provides consistent in vivo evidence across multiple tissues that BDNF deficiency is associated with enhanced NLRP3 inflammasome activation and coordinated alterations in autophagy- and ESCRT-III-related pathways, our mechanistic interpretation is primarily based on established protein markers (LC3B, SQSTM1/p62, and VPS4A). These markers are widely accepted indicators of pathway engagement but do not directly assess dynamic functional processes such as autophagic flux, membrane repair kinetics, or pyroptotic cell death. In addition, the use of a constitutive Bdnf +/− model may include developmental or compensatory adaptations. Nevertheless, the reproducible and tissue-wide nature of the findings strongly supports a regulatory role for BDNF in inflammasome control in vivo, and future studies incorporating functional assays and tissue-specific or inducible Bdnf models will further refine the underlying mechanisms.

5. Conclusions

BDNF deficiency enhances LPS plus nigericin-induced NLRP3 inflammasome activation and GSDMD-mediated pyroptosis. These effects are mediated through NF-κB activation and suppression of autophagy and ESCRT-III-dependent plasma membrane repair. Our findings highlight BDNF as a promising therapeutic target for controlling aberrant inflammasome activation (Figure 10).

Author Contributions

Conceptualization, Ş.E. and A.A.; methodology, Ş.E., N.S., E.Ş. and M.E.; software, Ş.E. and M.E.; validation, Ş.E. and A.A.; formal analysis, Ş.E. and M.E.; investigation, Ş.E. and M.E.; resources, Ş.E., İ.A. and A.A.; data curation, Ş.E. and A.A.; writing—original draft preparation, Ş.E. and M.E.; writing—review and editing, Ş.E., N.S., E.Ş., M.E., İ.A. and A.A.; visualization, Ş.E. and M.E.; supervision, A.A.; project administration, Ş.E. and A.A.; funding acquisition, A.A. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Scientific Research Projects Coordination Unit of Karadeniz Technical University, Turkey, grant numbers TDK-2021-9686 and TSA-2023-10700.

Institutional Review Board Statement

The animal study protocol was approved by Karadeniz Technical University Animal Experiments Local Ethics Committee (Protocol No: 2021/27, approved on 3 May 2021; Protocol No: 2023/5, approved on 15 February 2023).

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. A schematic representation of the LPS- and nigericin-induced inflammasome model, along with the experiments conducted on serum and tissue samples.
Figure 1. A schematic representation of the LPS- and nigericin-induced inflammasome model, along with the experiments conducted on serum and tissue samples.
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Figure 2. BDNF reduced NLRP3-dependent release of IL-1β and IL-18 cytokines. BDNF (A), IL-1β (B), and IL-18 (C) levels in serum samples. BDNF, IL-1β, and IL-18 levels were measured by ELISA. Data are presented as median (IQR), n = 10. Kruskal–Wallis test was used for overall group comparisons; pairwise comparisons were performed using the Mann–Whitney U test. * p < 0.05, ** p < 0.01, and *** p < 0.001.
Figure 2. BDNF reduced NLRP3-dependent release of IL-1β and IL-18 cytokines. BDNF (A), IL-1β (B), and IL-18 (C) levels in serum samples. BDNF, IL-1β, and IL-18 levels were measured by ELISA. Data are presented as median (IQR), n = 10. Kruskal–Wallis test was used for overall group comparisons; pairwise comparisons were performed using the Mann–Whitney U test. * p < 0.05, ** p < 0.01, and *** p < 0.001.
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Figure 3. BDNF reduced NLRP3 inflammasome activation and GSDMD-mediated pyroptosis through enhancing NF-κB inhibition in hippocampus tissues. NLRP3, active caspase-1 p20, GSDMD, and NF-κB p65 levels in hippocampus tissues were assessed by Western blot (A), and quantification of hippocampus NLRP3 (B), active caspase-1 p20 (C), GSDMD (D), and NF-κB p65 (E) protein levels normalized to loading controls. Data are expressed as mean ± S.E.M., n = 6. Kruskal–Wallis test was used for overall group comparisons; pairwise comparisons were performed using the Mann–Whitney U test. * p < 0.05, ** p < 0.01.
Figure 3. BDNF reduced NLRP3 inflammasome activation and GSDMD-mediated pyroptosis through enhancing NF-κB inhibition in hippocampus tissues. NLRP3, active caspase-1 p20, GSDMD, and NF-κB p65 levels in hippocampus tissues were assessed by Western blot (A), and quantification of hippocampus NLRP3 (B), active caspase-1 p20 (C), GSDMD (D), and NF-κB p65 (E) protein levels normalized to loading controls. Data are expressed as mean ± S.E.M., n = 6. Kruskal–Wallis test was used for overall group comparisons; pairwise comparisons were performed using the Mann–Whitney U test. * p < 0.05, ** p < 0.01.
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Figure 4. BDNF reduced NLRP3 inflammasome activation and GSDMD-mediated pyroptosis through enhancing NF-κB inhibition in cortex tissues. NLRP3, active caspase-1 p20, GSDMD, and NF-κB p65 levels in cortex tissues were assessed by Western blot (A), and quantification of cortex NLRP3 (B), active caspase-1 p20 (C), GSDMD (D), and NF-κB p65 (E) protein levels normalized to loading controls. Data are expressed as mean ± S.E.M., n = 6. Kruskal–Wallis test was used for overall group comparisons; pairwise comparisons were performed using the Mann–Whitney U test. * p < 0.05, ** p < 0.01.
Figure 4. BDNF reduced NLRP3 inflammasome activation and GSDMD-mediated pyroptosis through enhancing NF-κB inhibition in cortex tissues. NLRP3, active caspase-1 p20, GSDMD, and NF-κB p65 levels in cortex tissues were assessed by Western blot (A), and quantification of cortex NLRP3 (B), active caspase-1 p20 (C), GSDMD (D), and NF-κB p65 (E) protein levels normalized to loading controls. Data are expressed as mean ± S.E.M., n = 6. Kruskal–Wallis test was used for overall group comparisons; pairwise comparisons were performed using the Mann–Whitney U test. * p < 0.05, ** p < 0.01.
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Figure 5. BDNF reduced NLRP3 inflammasome activation and GSDMD-mediated pyroptosis through enhancing NF-κB inhibition in liver tissues. NLRP3, active caspase-1 p20, GSDMD, and NF-κB p65 levels in liver tissues were assessed by Western blot (A), and quantification of liver NLRP3 (B), active caspase-1 p20 (C), GSDMD (D), and NF-κB p65 (E) protein levels normalized to loading controls. Data are expressed as mean ± S.E.M., n = 6. Kruskal–Wallis test was used for overall group comparisons; pairwise comparisons were performed using the Mann–Whitney U test. * p < 0.05, ** p < 0.01.
Figure 5. BDNF reduced NLRP3 inflammasome activation and GSDMD-mediated pyroptosis through enhancing NF-κB inhibition in liver tissues. NLRP3, active caspase-1 p20, GSDMD, and NF-κB p65 levels in liver tissues were assessed by Western blot (A), and quantification of liver NLRP3 (B), active caspase-1 p20 (C), GSDMD (D), and NF-κB p65 (E) protein levels normalized to loading controls. Data are expressed as mean ± S.E.M., n = 6. Kruskal–Wallis test was used for overall group comparisons; pairwise comparisons were performed using the Mann–Whitney U test. * p < 0.05, ** p < 0.01.
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Figure 6. BDNF reduced NLRP3 inflammasome activation and GSDMD-mediated pyroptosis through enhancing NF-κB inhibition in epididymal adipose tissues. NLRP3, active caspase-1 p20, GSDMD, and NF-κB p65 levels in epididymal adipose tissues were assessed by Western blot (A), and quantification of epididymal adipose tissue NLRP3 (B), active caspase-1 p20 (C), GSDMD (D), and NF-κB p65 (E) protein levels normalized to loading controls. Data are expressed as mean ± S.E.M., n = 6. Kruskal–Wallis test was used for overall group comparisons; pairwise comparisons were performed using the Mann–Whitney U test. * p < 0.05, ** p < 0.01.
Figure 6. BDNF reduced NLRP3 inflammasome activation and GSDMD-mediated pyroptosis through enhancing NF-κB inhibition in epididymal adipose tissues. NLRP3, active caspase-1 p20, GSDMD, and NF-κB p65 levels in epididymal adipose tissues were assessed by Western blot (A), and quantification of epididymal adipose tissue NLRP3 (B), active caspase-1 p20 (C), GSDMD (D), and NF-κB p65 (E) protein levels normalized to loading controls. Data are expressed as mean ± S.E.M., n = 6. Kruskal–Wallis test was used for overall group comparisons; pairwise comparisons were performed using the Mann–Whitney U test. * p < 0.05, ** p < 0.01.
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Figure 7. BDNF reduced NLRP3 inflammasome activation and GSDMD-mediated pyroptosis through enhancing NF-κB inhibition in muscle tissues. NLRP3, active caspase-1 p20, GSDMD, and NF-κB p65 levels in muscle tissues were assessed by Western blot (A), and quantification of muscle tissue NLRP3 (B), active caspase-1 p20 (C), GSDMD (D), and NF-κB p65 (E) protein levels normalized to loading controls. Data are expressed as mean ± S.E.M., n = 6. Kruskal–Wallis test was used for overall group comparisons; pairwise comparisons were performed using the Mann–Whitney U test. * p < 0.05, ** p < 0.01.
Figure 7. BDNF reduced NLRP3 inflammasome activation and GSDMD-mediated pyroptosis through enhancing NF-κB inhibition in muscle tissues. NLRP3, active caspase-1 p20, GSDMD, and NF-κB p65 levels in muscle tissues were assessed by Western blot (A), and quantification of muscle tissue NLRP3 (B), active caspase-1 p20 (C), GSDMD (D), and NF-κB p65 (E) protein levels normalized to loading controls. Data are expressed as mean ± S.E.M., n = 6. Kruskal–Wallis test was used for overall group comparisons; pairwise comparisons were performed using the Mann–Whitney U test. * p < 0.05, ** p < 0.01.
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Figure 8. BDNF promotes SQSTM1/p62-dependent selective autophagy and LC3B-mediated autophagosome formation to inhibit NLRP3 inflammasome activation in hippocampus, cortex, liver, epididymal adipose, and muscle tissues. LC3B and SQSTM1/p62 levels in hippocampus were assessed by Western blot (A); quantification of hippocampus LC3B (B) and SQSTM1/p62 (C) protein levels normalized to loading controls. LC3B and SQSTM1/p62 levels in cortex tissues were assessed by Western blot (D); quantification of cortex tissues LC3B (E) and SQSTM1/p62 (F) protein levels normalized to loading controls. LC3B and SQSTM1/p62 levels in liver tissues were assessed by Western blot (G); quantification of liver tissues LC3B (H) and SQSTM1/p62 (I) protein levels normalized to loading controls. LC3B and SQSTM1/p62 levels in epididymal adipose tissues were assessed by Western blot (J); quantification of epididymal adipose tissues LC3B (K) and SQSTM1/p62 (L) protein levels normalized to loading controls. LC3B and SQSTM1/p62 levels in muscle tissues were assessed by Western blot (M); quantification of muscle tissues LC3B (N) and SQSTM1/p62 (O) protein levels normalized to loading controls. Data are expressed as mean ± S.E.M., n = 6. Kruskal–Wallis test was used for overall group comparisons; pairwise comparisons were performed using the Mann–Whitney U test. * p < 0.05, ** p < 0.01.
Figure 8. BDNF promotes SQSTM1/p62-dependent selective autophagy and LC3B-mediated autophagosome formation to inhibit NLRP3 inflammasome activation in hippocampus, cortex, liver, epididymal adipose, and muscle tissues. LC3B and SQSTM1/p62 levels in hippocampus were assessed by Western blot (A); quantification of hippocampus LC3B (B) and SQSTM1/p62 (C) protein levels normalized to loading controls. LC3B and SQSTM1/p62 levels in cortex tissues were assessed by Western blot (D); quantification of cortex tissues LC3B (E) and SQSTM1/p62 (F) protein levels normalized to loading controls. LC3B and SQSTM1/p62 levels in liver tissues were assessed by Western blot (G); quantification of liver tissues LC3B (H) and SQSTM1/p62 (I) protein levels normalized to loading controls. LC3B and SQSTM1/p62 levels in epididymal adipose tissues were assessed by Western blot (J); quantification of epididymal adipose tissues LC3B (K) and SQSTM1/p62 (L) protein levels normalized to loading controls. LC3B and SQSTM1/p62 levels in muscle tissues were assessed by Western blot (M); quantification of muscle tissues LC3B (N) and SQSTM1/p62 (O) protein levels normalized to loading controls. Data are expressed as mean ± S.E.M., n = 6. Kruskal–Wallis test was used for overall group comparisons; pairwise comparisons were performed using the Mann–Whitney U test. * p < 0.05, ** p < 0.01.
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Figure 9. BDNF activated the ESCRT-III-mediated plasma membrane repair system to prevent pyroptosis in hippocampus, cortex, liver, epididymal adipose, and muscle tissues. VPS4A levels in the hippocampus were assessed by Western blot (A), and quantification of hippocampus VPS4A (B) protein levels normalized to loading controls. VPS4A levels in cortex tissues were assessed by Western blot (C); quantification of cortex tissues’ VPS4A (D) protein levels normalized to loading controls. VPS4A levels in liver tissues were assessed by Western blot (E); quantification of liver tissues’ VPS4A (F) protein levels normalized to loading controls. VPS4A levels in epididymal adipose tissues were assessed by Western blot (G); quantification of epididymal adipose tissues’ VPS4A (H) protein levels normalized to loading controls. VPS4A levels in muscle tissues were assessed by Western blot (I); quantification of muscle tissues’ VPS4A (J) protein levels normalized to loading controls. Data are expressed as mean ± S.E.M., n = 6. Kruskal–Wallis test was used for overall group comparisons; pairwise comparisons were performed using the Mann–Whitney U test. * p < 0.05, ** p < 0.01.
Figure 9. BDNF activated the ESCRT-III-mediated plasma membrane repair system to prevent pyroptosis in hippocampus, cortex, liver, epididymal adipose, and muscle tissues. VPS4A levels in the hippocampus were assessed by Western blot (A), and quantification of hippocampus VPS4A (B) protein levels normalized to loading controls. VPS4A levels in cortex tissues were assessed by Western blot (C); quantification of cortex tissues’ VPS4A (D) protein levels normalized to loading controls. VPS4A levels in liver tissues were assessed by Western blot (E); quantification of liver tissues’ VPS4A (F) protein levels normalized to loading controls. VPS4A levels in epididymal adipose tissues were assessed by Western blot (G); quantification of epididymal adipose tissues’ VPS4A (H) protein levels normalized to loading controls. VPS4A levels in muscle tissues were assessed by Western blot (I); quantification of muscle tissues’ VPS4A (J) protein levels normalized to loading controls. Data are expressed as mean ± S.E.M., n = 6. Kruskal–Wallis test was used for overall group comparisons; pairwise comparisons were performed using the Mann–Whitney U test. * p < 0.05, ** p < 0.01.
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Figure 10. Schematic representation of the relationship between BDNF deficiency and NLRP3 inflammasome activation. The red line is the inhibition, and the green line is the activation.
Figure 10. Schematic representation of the relationship between BDNF deficiency and NLRP3 inflammasome activation. The red line is the inhibition, and the green line is the activation.
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MDPI and ACS Style

Erdem, Ş.; Sağlam, N.; Şahin, E.; Erdem, M.; Abidin, İ.; Alver, A. Brain-Derived Neurotrophic Factor Deficiency Exacerbates Innate Immune Responses by Enhancing NLRP3 Inflammasome Activation and GSDMD-Mediated Pyroptosis in Mice. Medicina 2026, 62, 384. https://doi.org/10.3390/medicina62020384

AMA Style

Erdem Ş, Sağlam N, Şahin E, Erdem M, Abidin İ, Alver A. Brain-Derived Neurotrophic Factor Deficiency Exacerbates Innate Immune Responses by Enhancing NLRP3 Inflammasome Activation and GSDMD-Mediated Pyroptosis in Mice. Medicina. 2026; 62(2):384. https://doi.org/10.3390/medicina62020384

Chicago/Turabian Style

Erdem, Şeniz, Neslihan Sağlam, Elif Şahin, Mehmet Erdem, İsmail Abidin, and Ahmet Alver. 2026. "Brain-Derived Neurotrophic Factor Deficiency Exacerbates Innate Immune Responses by Enhancing NLRP3 Inflammasome Activation and GSDMD-Mediated Pyroptosis in Mice" Medicina 62, no. 2: 384. https://doi.org/10.3390/medicina62020384

APA Style

Erdem, Ş., Sağlam, N., Şahin, E., Erdem, M., Abidin, İ., & Alver, A. (2026). Brain-Derived Neurotrophic Factor Deficiency Exacerbates Innate Immune Responses by Enhancing NLRP3 Inflammasome Activation and GSDMD-Mediated Pyroptosis in Mice. Medicina, 62(2), 384. https://doi.org/10.3390/medicina62020384

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