Differential Cytokine Regulation in Microglial Endotoxin Tolerance

Abstract

Background: Endotoxin tolerance describes the phenomenon whereby prior lipopolysaccharide (LPS) exposure attenuates inflammatory responses to subsequent LPS challenge. Studies have reported the involvement of different mediators of the toll-like receptor (TLR)-4 signaling pathway in endotoxin tolerance. Methods: We first examined dose- and time-dependent production of cytokines following LPS treatment and then examined cytokine production in BV2 cells pretreated with 5 ng/mL LPS for 24 h, followed by secondary challenge with 1 µg/mL LPS for four hours. To examine which inflammatory cytokine could induce tolerance, we pretreated BV2 cells with 1 µg/mL IL-1β, IL-6, or TNF-α for 24 h, followed by secondary challenge with 1 μg/mL LPS for four hours, and then examined cytokine production by ELISA. Results: Our data showed that LPS induced dose- and time-dependent production of IL-1β, IL-6, and TNF-α. Pretreatment with 5 ng/mL LPS significantly reduced the production of IL-1β and TNF-α in response to secondary challenge, while IL-6 production was slightly enhanced. We also found that pretreatment with IL-1β did not attenuate production of TNF-α but slightly enhanced IL-6 following secondary challenge with 1 µg/mL LPS. In contrast, pretreatment with IL-6 or TNF-α significantly attenuated subsequent LPS-induced IL-1β production without affecting the production of the other. Conclusions: Endotoxin tolerance in BV2 microglial cells selectively suppresses IL-1β and TNF-α while preserving IL-6 production. Both IL-6 and TNF-α independently induce tolerance specifically to IL-1β, suggesting negative feedback regulations. These findings reveal that endotoxin tolerance involves selective rather than global suppression of inflammatory mediators and cross-regulation between LPS and cytokine-induced signaling pathways.

1. Introduction

Microglia, the resident immune cells of the Central Nervous System (CNS), constitutively express a range of pattern recognition receptors (PRRs), including toll-like receptors (TLRs), that bind to exogenous pathogen-associated molecular patterns (PAMPs) or endogenous damage-associated molecular patterns (DAMPs) [1]. They act as sensors that actively survey the brain. Upon detection of PAMPs or DAMPs, microglia become activated, orchestrating an inflammatory response [2]. Cluster of Differentiation 14 (CD14) transfers endotoxin, lipopolysaccharide (LPS), to TLR-4/MD-2 (Myeloid Differentiation factor 2) complex [3], leading to the activation of Myeloid differentiation factor 88 (MyD88)-dependent and MyD88-independent pathways and multiple signaling components including nuclear factor (NF)-κB and interferon regulatory factor (IRF)-3, and the subsequent production of pro-inflammatory cytokines including tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 [4].

In addition to recognition of extracellular LPS by TLR-4 and CD14, intracellular LPS binds to the cytosolic proteases, caspase-4 and caspase-5 in humans and caspase-11 in mice, leading to the formation of a non-canonical inflammasome that, together with guanylate-binding proteins (GBPs), facilitates the assembly and activation of the NLRP3 (NOD-, LRR- and pyrin domain-containing protein 3) inflammasome [5]. Activation of this pathway initiates pyroptosis, a lytic, pro-inflammatory form of programmed cell death [6]. Together with TLR-4-mediated signaling, these innate immune pathways drive pro-inflammatory cascades directed at bacterial eradication. However, dysregulated activation of either pathway has been implicated in the pathogenesis of both acute and chronic inflammatory conditions [7].

Pretreatment of an organism or cell with LPS has been found to attenuate the response to subsequent challenge, which is termed endotoxin tolerance [8]. It is a complex, regulated response marked by decreased pro-inflammatory cytokines, increased anti-inflammatory mediators, and increased phagocytic ability upon subsequent challenge with LPS. For example, repeated exposure to a low dose of LPS reduces the production of IL-6 and TNF-α and liver injury in rat models of acute liver failure [9]. Endotoxin tolerance also attenuates plasma TNF-α and IL-6 and augments bacterial clearance in both blood and peritoneal fluid in a murine model of polymicrobial sepsis induced by cecal ligation and puncture [10]. LPS-tolerant mice exhibit a diminished serum interferon (IFN)-γ response to subsequent challenge with Staphylococcus aureus [11].

Studies have reported that endotoxin tolerance is associated with inhibition of TLR-4 signaling pathways. For example, endotoxin tolerance is associated with decreased CD14 mRNA expression, reduced TLR-4-MyD88 complex formation, impairment of interleukin-1 receptor-associated kinase (IRAK)-1 activity, and defects in the activation of mitogen-activated protein kinases (MAPKs) and nuclear factor (NF)-κB in human mono mac 6 cells and RAW 264.7 cells [12]. Knockout of negative regulators of the TLR-4 signaling pathway, such as IRAK-M and MAPK Phosphatase 1 (MKP-1), is reported to abolish endotoxin tolerance in mouse bone marrow-derived macrophages and THP1 cells [13,14]. LPS treatment has also been shown to increase the expression of microRNA such as miR146 and miR155 to attenuate TLR-4 signaling through the regulation of IRAK-1 and TNF receptor-associated factor (TRAF)-6 proteins in THP1 cells and in mouse bone-derived macrophages, respectively [15,16].

In this study, we first examined dose- and time-dependent LPS induction of IL-1β, IL-6, or TNF-α in BV2 cells. We then pretreated BV2 cells with vehicle or 5 ng/mL LPS or 1 µg/mL cytokine, including IL-1β, IL-6, or TNF-α, for 24 h, followed by secondary challenge with 1 μg/mL LPS for 4 hours to investigate endotoxin tolerance and cytokine-specific effects. This study was designed to better understand inflammatory signaling in microglia, which may potentially shed light on the regulation of inflammatory responses in the CNS and provide new treatment options for dysregulated neuroinflammation.

2. Materials and Methods

2.1. Cell Culture

BV2 cells (kindly provided by Dr. Jau-Shyong Hong from the National Institute of Health) were grown in Dulbecco’s Modified Eagle’s Medium (DMEM; HyClone Laboratories, Logan, UT, USA) supplemented with 10% Fetal Calf Serum (FCS; ThermoFisher Scientific, Waltham, MA, USA) at 37 °C in a humidified incubator with 5% CO₂. To examine time- and dose-dependent effects of LPS on cell viability, BV2 cells were seeded in 96-well cell culture plates at a density of 4000 cells per well and allowed to adhere overnight. Then, the cell culture media were replaced with serum-free media, and cells were treated with various concentrations of LPS for different periods of time. To examine time- and dose-dependent induction of cytokines following LPS treatment, cells were seeded in 24-well cell culture plates at a density of 20,000 cells per well. After overnight incubation, culture media were replaced with serum-free media and treated with different concentrations of LPS for different periods of time. For endotoxin tolerance experiments, cells were seeded in 24-well cell culture plates at a density of 20,000 cells per well. After overnight incubation, culture media were replaced with serum-free media and treated with vehicle, 5 ng/mL LPS, or 1 µg/mL cytokine, TNF-α, IL-1β, or IL-6, for 24 hours. Then cell culture media were removed and replaced with fresh serum-free media. Cells were then stimulated with vehicle or 1 µg/mL LPS for four hours. Each treatment has three replicates. Each experiment was repeated three times.

• Preparation of LPS and cytokines

LPS derived from Escherichia coli O55:B5 (Sigma-Aldrich, St. Louis, MO, USA) was used in all experiments. It was dissolved in sterile phosphate-buffered saline (PBS) to prepare a stock solution of 1 mg/mL, aliquoted, and stored at −80 °C until use. Recombinant carrier-free mouse IL-1β, IL-6, and TNF-α proteins (with less than 0.01 endotoxin unit per 1 μg of the protein by the Limulus Amebocyte Lysate (LAL) method and greater than 97% purity by SDS-PAGE under reducing conditions and visualized by silver stain) were purchased from R&D Systems (Minneapolis, MN, USA), reconstituted in sterile PBS at 100 µg/mL, aliquoted, and stored at −80 °C until use.

• MTT Assay

At the end of treatment, cell viability was assessed using the MTT assay as previously described [17]. Briefly, 10 µL of 5 mg/mL MTT solution (Sigma-Aldrich, St. Louis, MO, USA) was added to each well two hours prior to the end of treatment and incubated for an additional two hours. Following incubation, the resulting formazan crystals were solubilized with the addition of 100 µL solubilization solution, and absorbance at 570 nm was measured using a Varioskan LUX Multimode Microplate Reader (ThermoFisher Scientific, Waltham, MA, USA). Cell viability was expressed as a percentage relative to vehicle-treated control cells.

• ELISA

At the end of treatment, cell culture media were collected for ELISA assay to analyze the production of IL-6 and TNF-α by BV2 cells. Cells in each well were lysed with 200 µL of RIPA buffer (Sigma-Aldrich, Saint Louis, MO, USA) supplemented with 1× protease inhibitor (Roche, San Francisco, CA, USA) for 30 minutes at 4 °C. Cell lysates were collected and used for the ELISA assay of IL-1β. IL-1β level was normalized against total protein concentration of the cell lysate as measured using the Pierce™ BCA protein assay kit (ThermoFisher Scientific, Rockford, IL, USA). ELISA kits were purchased from Biolegend (San Diego, CA, USA), and the ELISA was performed following the manufacturer’s instructions.

2.2. Semi-Quantitative Reverse-Transcription Polymerase Chain Reaction (RT-PCR)

At the end of treatment, BV2 cells were lysed with TRI Reagent^® (Molecular Research Center, Cincinnati, OH, USA) and total RNA was extracted according to the manufacturer’s instructions. RNA was reverse transcribed into cDNA, which was subsequently amplified by semi-quantitative RT-PCR using gene-specific sense and antisense primers for mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a housekeeping gene for normalization, as well as TLR-4 and CD14. PCR reactions were performed as previously described [18]. The primers were synthesized by Eurofins Genomics (Louisville, KY, USA), and their sequences were as follows: GAPDH sense, 5′-GGAGAAACCTGCCAAGTATGA-3′ and antisense, 5′-CCTGTTGCTGTAGCCGTATT-3′; TLR-4 sense, 5′-GGCAGCAGGTGGAATTGTAT-3′ and antisense, 5′-CTTAGCAGCCATGTGTTCCA-3′; CD14 sense, 5′-CTGATCTCAGCCCTCTGTCC-3′ and antisense, 5′-GCTTCAGCCCAGTGAAAGAC-3′. The PCR reactions were heated to 94 °C for 5 minutes, followed by appropriate cycles of 30 s at 94 °C, 30 s at 57 °C, and 30 s at 72 °C, followed by a 7-minute extension at 72 °C after the final cycle. GAPDH, TLR-4, and CD14 were amplified for 25, 27, and 25 cycles, respectively. PCR products were then separated using 2.0% agarose gel electrophoresis, and images were captured using a UVP GelDoc-It™ imaging system (UVP, Upland, CA, USA). Band intensities were then quantified and normalized to GAPDH expression levels within the same sample.

2.3. Statistical Analysis

Statistical analysis was performed using Prism version 6.0 (GraphPad Software, San Diego, CA, USA). For dose–response and time course studies, statistical analysis was performed using one-way ANOVA followed by Dunnett’s post-test. For tolerance studies, statistical analysis was performed using a two-way ANOVA followed by Tukey’s multiple comparisons test.

3. Results

3.1. Dose-Dependent and Time-Dependent Effects of LPS on the Viability of BV2 Cells

To investigate the dose-dependent effects of LPS on cell viability, BV2 cells were exposed to a range of LPS concentrations (0–1000 ng/mL) for 24 hours in serum-free media, and cell viability was assessed by MTT assay. As shown in Figure 1A, cell viability at 5 ng/mL was comparable to that of the untreated control. However, cell viability decreased to approximately 60% following 24-hour treatment with 10 ng/mL LPS and remained consistently reduced across higher concentrations up to 1000 ng/mL LPS (p < 0.001).

To investigate the time-dependent effects of LPS on cell viability, cells were exposed to either 5 ng/mL or 1 µg/mL LPS for 0, 4, 24, and 48 hours, and cell viability was assessed by MTT assay. As shown in Figure 1B, treatment with 5 ng/mL LPS did not significantly affect cell viability at 24 hours; however, cell viability decreased significantly after 48-hour treatment with 5 ng/mL LPS (p < 0.001). As shown in Figure 1C, treatment with 1 µg/mL LPS had no significant effects on cell viability at 4 hours, but caused a steep decline at 24 hours, with cell viability further reduced to approximately 13% by 48 hours.

3.2. Dose-Dependent Production of Inflammatory Cytokines in LPS-Stimulated BV2 Cells

BV2 microglial cells were treated with increasing concentrations of LPS from 0 to 1000 ng/mL for four hours to evaluate dose-dependent production of cytokines. All three cytokines exhibited significant dose-dependent increases (Figure 2). IL-1β production was minimal in control cells but increased significantly starting at 1 ng/mL LPS (p < 0.01) and further increased at 5 ng/mL and above (p < 0.001), with IL-1β level plateauing at around 500–1000 ng/mL LPS (Figure 2A). IL-6 demonstrated a similar dose–response pattern, with significant elevation beginning at 5 ng/mL (p < 0.05) and progressive increases through 500–1000 ng/mL (Figure 2B). TNF-α showed minimal production at 1 ng/mL LPS but exhibited significant induction starting at 5 ng/mL (p < 0.001). The robust production of TNF-α continued to 1000 ng/mL LPS (Figure 2C).

3.3. Time-Dependent Inflammatory Response to LPS Treatment

BV2 cells were treated with 5 ng/mL or 1 μg/mL LPS for varying durations up to 8 hours. At 5 ng/mL LPS, IL-1β production became significantly elevated by two hours (p < 0.001) and continued to increase through eight hours (Figure 3A). IL-6 levels showed a progressive time-dependent increase, with significant elevation at 4 hours (p < 0.001) and continued to increase at 6–8 hours post-treatment (Figure 3B). TNF-α production followed a similar pattern, with significant increases detectable by two hours (p < 0.001) and sustained elevation through eight hours post-treatment (Figure 3C). When cells were treated with 1 μg/mL LPS, the kinetics of cytokine production were similar to those of 5 ng/mL LPS, but with an earlier onset for some cytokines (Figure 3D–F). Both IL-1β and IL-6 showed significant elevation by two hours, and TNF-α showed significant elevation by one hour post-treatment (p < 0.001). All three cytokines continued to increase through eight hours of treatment. These findings demonstrate that LPS induced a robust dose- and time-dependent inflammatory response in BV2 microglial cells.

3.4. LPS-Induced Production of Cytokines in Microglia Previously Exposed to Low-Dose LPS

To investigate whether prior exposure to low-dose LPS could attenuate subsequent inflammatory response to high-dose LPS, BV2 cells were pretreated with either vehicle or 5 ng/mL LPS for 24 h, followed by secondary stimulation with vehicle or 1 μg/mL LPS for 4 hours (Figure 4). As expected, cells receiving vehicle pretreatment followed by LPS stimulation (V+L) exhibited robust production of all three pro-inflammatory cytokines. Cells pretreated with LPS and stimulated with vehicle (L+V) showed minimal production of IL-1β, a low level of TNF-α, but a high level of IL-6 production compared with V+V. Cells pretreated with LPS for 24 hours prior to secondary LPS challenge (L+L) demonstrated a differential pattern of endotoxin tolerance across the three cytokines. IL-1β production in the L+L group was significantly reduced as compared to the V+L group (Figure 4A). Similarly, TNF-α production was significantly attenuated in the L+L group as compared to V+L (Figure 4C). In contrast, IL-6 production in the L+L group remained robust, showing no attenuation and even a slight increase compared to the V+L group (Figure 4B). These findings reveal that LPS pretreatment induced cytokine-specific endotoxin tolerance in BV2 microglial cells, with IL-1β and TNF-α responses being suppressed while IL-6 production was preserved or enhanced following secondary LPS challenge.

3.5. IL-1β Pretreatment Did Not Decrease Subsequent LPS-Induced IL-6 and TNF-α Production

To determine whether pretreatment with the pro-inflammatory cytokine IL-1β could modulate subsequent responses to LPS, BV2 cells were pretreated with either vehicle or 1 μg/mL recombinant IL-1β for 24 h, followed by stimulation with vehicle or 1 μg/mL LPS for four hours (Figure 5). V+L cells showed robust production of both IL-6 and TNF-α (p < 0.001). IL-1β pretreatment alone (IL-1β+V) did not significantly elevate either cytokine above their baseline levels in vehicle-treated controls (V+V). Cells pretreated with IL-1β for 24 hours prior to LPS challenge (IL-1β+L) exhibited significantly enhanced production of IL-6 and TNF-α compared to V+V cells (p < 0.001). IL-6 level in the IL-1β+L group increased over the V+L group (Figure 5A). TNF-α production in the IL-1β+L group was comparable to that in the V+L group (Figure 5B). These findings demonstrate that IL-1β pretreatment did not attenuate the inflammatory response of BV2 microglial cells to subsequent LPS stimulation.

3.6. IL-6 Pretreatment Decreased Subsequent LPS-Induced IL-1β but Not TNF-α Production

To determine the effects of IL-6 pretreatment on LPS-induced inflammatory responses, BV2 cells were pretreated with vehicle or 1 μg/mL IL-6 for 24 hours, followed by stimulation with vehicle or 1 μg/mL LPS for four hours. As shown in Figure 6, LPS stimulation markedly increased IL-6 and TNF-α protein levels compared to vehicle control. Pretreatment with IL-6 alone (IL-6+V) did not significantly alter IL-1β levels compared to vehicle control. However, IL-6 pretreatment significantly attenuated LPS-induced elevation of IL-1β in IL-6+L cells (Figure 6A). IL-6 pretreatment alone had no significant effect on the level of TNF-α (IL-6+V) as compared to V+V. The protein level of TNF-α in IL-6+L cells was comparable to that in V+L cells. These findings suggest that IL-6 pretreatment selectively modulates certain aspects of the LPS-induced inflammatory response in BV2 cells, specifically attenuating IL-1β but not TNF-α production.

3.7. TNF-α Pretreatment Decreased Subsequent LPS-Induced IL-1β but Not IL-6 Production

To investigate the effect of TNF-α pretreatment on LPS-induced inflammatory cytokine production, BV2 cells were pretreated with vehicle or 1 μg/mL TNF-α for 24 h, followed by stimulation with vehicle or 1 μg/mL LPS for four hours. As shown in Figure 7A, TNF-α pretreatment alone (TNF-α+V) did not significantly affect the production of IL-1β compared to vehicle control. TNF-α pretreatment significantly reduced the LPS-induced IL-1β response as compared to V+L cells, indicating an attenuating effect on IL-1β production (Figure 7A). In contrast, TNF-α pretreatment minimally affected IL-6 levels in TNF-α+L cells as compared to the V+L group (Figure 7B). These findings demonstrate that TNF-α pretreatment exerts divergent modulatory effects on LPS-induced cytokine responses in BV2 cells, suppressing IL-1β production without attenuating the production of IL-6.

3.8. Expression of TLR-4 and CD14 Following LPS Treatment

To examine the expression levels of TLR4 and CD14 during endotoxin tolerance, BV2 cells were pretreated with vehicle or 5 ng/mL LPS for 24 hours, followed by stimulation with vehicle or 1 µg/mL LPS for 4 hours. RNA was then extracted, and the expression of TLR-4 and CD14 was assessed by semi-quantitative RT-PCR. As shown in Figure 8A & B, TLR-4 mRNA expression was modestly elevated in cells pretreated with vehicle and stimulated with LPS (V+L) compared to cells pretreated with vehicle and stimulated with vehicle (V+V), whereas the mRNA level of TLR-4 in L+V and L+L groups was not significantly different from V+V or V+L groups. As shown in Figure 8A & C, CD14 mRNA expression in the V+L group did not differ significantly among any of the groups tested, although a modest increase was observed in the V+L group relative to the V+V control.

4. Discussion

The present study investigated the role of cytokines in the development of endotoxin tolerance in BV2 microglial cells, with a particular focus on the role of individual pro-inflammatory cytokines in modulating subsequent inflammatory responses. Our findings demonstrate that LPS induced a robust, dose- and time-dependent production of IL-1β, IL-6, and TNF-α in BV2 cells. Since 5 ng/mL LPS was found to induce significant production of IL-1β, IL-6 and TNF-α without significantly affecting cell viability, it was used as the low-dose pretreatment to induce tolerance. Treatment with 1 µg/mL LPS was able to induce a significant increase in all three cytokines at four hours post-treatment without significantly affecting cell viability, therefore, it was used as the secondary challenge dose to test how prior exposure to a low dose LPS affected the cells’ cytokine response to high dose LPS. We observed that prior LPS exposure induced cytokine-specific endotoxin tolerance, with IL-1β and TNF-α responses being significantly suppressed while IL-6 production remained preserved or even enhanced following secondary LPS challenge. Selective tolerance has also been observed in other immune cell types and is thought to reflect the immune system’s attempt to maintain certain protective functions while limiting excessive inflammation [19]. Furthermore, pretreatment with individual cytokines revealed differential modulatory effects. Both IL-6 and TNF-α pretreatment selectively attenuated subsequent LPS-induced IL-1β production, while IL-1β pretreatment failed to induce tolerance to the production of IL-6 and TNF-α.

IL-1β has been implicated in perpetuating immune responses and contributing to the development of a range of CNS conditions, including multiple sclerosis, traumatic brain injury, diabetic retinopathy, and Alzheimer’s disease [20,21]. TNF-α is a pleiotropic mediator of diverse physiological and neurological functions, including normal regulatory roles and immune responses to infectious agents [22,23]. In the inflamed or diseased brain, TNF-α can potentiate glutamate-mediated cytotoxicity by inhibiting glutamate transport in astrocytes and directly triggering surface expression of calcium-permeable AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) and NMDA (N-methyl-D-aspartate) receptors on neurons, while decreasing inhibitory gamma-aminobutyric acid (GABA)-A receptors [22]. In this context, the suppression of both TNF-α and IL-1β during endotoxin tolerance may represent a coordinated neuroprotective regulation that limits excitotoxic and neuroinflammatory damage in the CNS during repeated inflammatory challenges.

In our studies, BV2 microglia cells retained the capacity to produce IL-6 during endotoxin tolerance while limiting the production of IL-1β and TNF-α. IL-6 has both pro-inflammatory and anti-inflammatory properties with both neuroprotective and potentially detrimental effects depending on the context and duration of exposure [24,25]. It has been shown to play critical roles in tissue repair and regeneration [26,27]. Acute IL-6 has been shown to promote neuronal survival and tissue repairs mediated through Janus kinase (JAK) and signal transducer and activator of transcription (STAT)-3 pathway. Chronically elevated IL-6 has been associated with astrogliosis and blood–brain barrier disruption in neurological disease [24]. The balance between the opposing roles of IL-6 may therefore determine whether the preservation of its production during endotoxin tolerance is ultimately neuroprotective or detrimental in the context of CNS pathologies.

Previous studies have identified several mechanisms that contribute to endotoxin tolerance, including downregulation of TLR-4 expression, upregulation of negative regulators such as IRAK-M and MKP-1, increased expression of suppressive microRNAs, and epigenetic modifications that alter the accessibility of inflammatory gene promoters [28,29,30]. Given the critical roles of TLR-4 and CD14 in LPS-induced inflammatory pathways [31], we examined their mRNA expression using semi-quantitative PCR and found that their expression levels were not decreased in endotoxin-tolerant cells, suggesting that the attenuated IL-1β and TNF-α production observed in endotoxin-tolerant cells was not attributable to the downregulation of these receptors. Future work employing real-time PCR and specific TLR-4 or CD14 inhibitors may provide further mechanistic insight into receptor-level contributions to endotoxin tolerance induction.

Besides activating TLR-4 signaling, LPS also elevates reactive oxygen species and reduces mitochondrial membrane potential [32], implicating mitochondrial stress in endotoxin tolerance through mitohormesis, an adaptive mitochondrial stress response in which mitochondrial reactive oxygen and electrophilic species act as TLR-dependent signals that impair pro-inflammatory gene transcription as the cells transition to an LPS-tolerant state, serving as a negative feedback mechanism to restrain inflammation [33].

To investigate which cytokines contribute to the establishment of endotoxin tolerance, we pretreated BV2 cells with recombinant IL-1β, IL-6, or TNF-α before LPS challenge. Interestingly, both IL-6 and TNF-α pretreatment significantly attenuated subsequent LPS-induced IL-1β production even though they did not affect the production of each other following subsequent LPS stimulation, suggesting that these cytokines can initiate tolerance regulations that specifically target IL-1β responses. This is particularly noteworthy as IL-1β is a potent pro-inflammatory cytokine that plays a central role in neuroinflammation and has been implicated in the pathogenesis of various neurodegenerative diseases [21]. It is possible that IL-6 and TNF-α pretreatment may induce the expression of negative regulators that specifically interfere with IL-1β gene transcription or processing. Alternatively, these cytokines may activate anti-inflammatory signaling pathways that selectively antagonize the signals required for IL-1β production. These areas may warrant further investigation.

Other studies have also examined cross-regulation of cytokine production. Ferlito et al. have demonstrated that TNF-α and LPS similarly induce phosphorylation of downstream mediators, including extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), and promote DNA binding of p50/p65 NF-κB heterodimer in THP-1 cells. They further showed that TNF-α and LPS exert differential effects on IRAK in that IRAK degradation is induced by LPS but unaffected by TNF-α [34]. Park et al. have reported that TNF-induced cross-tolerance may be mediated by suppression of LPS-induced signaling and chromatin remodeling in macrophages [35]. It would be interesting to further examine how downstream signaling is differentially affected by LPS and different cytokines in microglial cells during endotoxin tolerance.

In our study, we examined the protein levels of TNF-α, IL-6, and IL-1β. It should be noted that the expression of TNF-α and IL-6 is regulated at both the transcriptional and post-transcriptional levels [36,37], and IL-1β not only undergoes transcriptional and post-transcriptional regulation but also requires inflammasome activation and caspase-1-mediated cleavage of its inactive precursor pro-IL-1β into its biologically active secreted form [38]. Therefore, mRNA and protein levels may not always correlate linearly for these cytokines. Nevertheless, our current data measuring protein levels of IL-1β in cell lysates and IL-6 and TNF-α in culture media provide direct evidence of cytokine production at the protein level during endotoxin tolerance. Future studies examining the mRNA levels of these cytokines would provide complementary information on their transcriptional regulation and further illuminate the multilayered mechanisms governing their expression under the conditions tested.

We used the BV2 mouse microglial cell line in our study. While it is a widely used model for studying microglial biology and neuroinflammation, it carries several limitations. Firstly, BV2 cells may not faithfully represent the inflammatory responses of primary mouse microglia [39,40]. Secondly, given their murine origin, they do not fully recapitulate the transcriptomic and immunological characteristics of human microglia [41,42]. Thirdly, while both murine microglia and human microglial HMC3 cells have been shown to produce pro-inflammatory cytokines in response to LPS [43,44], humans exhibit greater sensitivity to LPS than rodents, potentially due to differences in caspase expression. Specifically, humans express caspase-4 and caspase-5, whereas mice rely on caspase-11, to regulate caspase-1 activity and mediate endotoxin-induced IL-1β release [45]. These species-specific differences in inflammatory signaling may influence the extent to which findings in BV2 cells are directly translatable to human disease contexts. Consequently, validation of the present findings in human microglial models will be an important direction for future studies before their relevance to human neuroinflammatory and neurodegenerative diseases can be fully established.

Our findings have important implications for understanding the regulation of neuroinflammation. Microglia play central roles in both the initiation and resolution of inflammatory responses in the CNS, and dysregulated microglial activation has been implicated in numerous neurological disorders, including Alzheimer’s disease, Parkinson’s disease, multiple sclerosis, and stroke [21,46]. The ability of microglia to develop endotoxin tolerance and to selectively regulate different cytokine responses may represent an important strategy for preventing excessive or prolonged inflammation in the brain.

5. Conclusions

In conclusion, our study demonstrates that endotoxin tolerance in BV2 microglial cells is characterized by selective suppression of IL-1β and TNF-α responses while IL-6 production is preserved. We further show that pretreatment with IL-6 or TNF-α can selectively attenuate subsequent LPS-induced IL-1β production, suggesting that these cytokines may participate in negative feedback regulation of IL-1β responses. These findings provide new insights into the complex regulatory networks that control microglial cytokine production and suggest that endotoxin tolerance involves selective rather than global suppression of inflammatory responses. Understanding cytokine-specific regulations during endotoxin tolerance may enable targeted therapeutic strategies to modulate neuroinflammation while preserving beneficial immune functions.