MicroRNA-132 Negatively Regulates Palmitate-Induced NLRP3 Inflammasome Activation through FOXO3 Down-Regulation in THP-1 Cells

Saturated fatty acids were proposed to activate the NLRP3 inflammasome, a molecular platform that mediates the processing of interleukin (IL)-1β and IL-18. However, the mechanisms underlying the miRNA-mediated regulation of palmitate (PA)-induced inflammasome activation are unclear. We examined the role of miR-132 in PA-induced NLRP3 inflammasome activation in THP-1 cells. To understand the regulatory role of miR-132 in inflammasome activation, we either overexpressed or suppressed miR-132 in THP-1 cells that expressed the NLRP3 inflammasome in response to stimulation by PA. We analyzed the mRNA and protein levels of NLRP3, caspase-1 p10, IL-18, and IL-1β; caspase-1 activity; and IL-1β secretion. The presence of PA activated the NLRP3 inflammasome and increased miR-132 expression. Overexpression of miR-132 reduced caspase-1 p10, IL-18, and IL-1β, while the suppression of miR-132 enhanced inflammasome activation. In addition, miR-132 regulated the mRNA and protein expression of FOXO3, which is a potential target of miR-132 in these cells. FOXO3 suppression by small interfering RNA decreased NLRP3 inflammasome activity stimulated by PA. Knockdown of FOXO3 attenuated NLRP3 inflammasome activation by the miR-132 inhibitor. Based on these findings, we conclude that miR-132 negatively regulates PA-induced NLRP3 inflammasome activation through FOXO3 down-regulation in THP-1 cells.


Introduction
Obesity and its comorbidities, including type 2 diabetes and cardiovascular disease, are associated with a state of chronic low-grade inflammation, characterized by up-regulated cytokine production and inflammation [1]. Inflammation is an innate immune response to repair tissue injury, and is predominantly mediated via myeloid cells such as monocytes, macrophages, and neutrophils. Inflammation is triggered by pathogen-associated molecular patterns and endogenous substances that are released during tissue or cell damage (danger-associated molecular patterns) [2,3]. The inflammasome is an important part of our innate immune system that responds to danger signals that are sensed by many different NOD-like receptors (NLRs) [4]. Inflammasomes are molecular platforms that modulate innate immune functions by activating caspase-1 and by catalyzing the proteolytic processing and secretion of interleukin (IL)-1β and IL-18 [5]. To date, nucleotide-binding 4 × 10 5 cells per well and incubated for 2-24 h in cell culture media containing different concentrations of PA. Control cells were incubated with the same medium containing the same amount of solvent (BSA) used to dissolve the PA.
Peritoneal exudate cells obtained from C57BL/6 male mice were washed twice and resuspended in the same medium as above. Macrophages were isolated from the peritoneal exudate cells as described previously [17]. The peritoneal exudate cells were seeded on Teflon-coated petri dishes (100 × 15 mm) at densities of 5-6 × 10 5 cells/cm 2 , and the macrophages were allowed to adhere for 2-3 h at 37 • C in a 5% CO 2 humidified atmosphere. The non-adherent cells were removed by washing the dishes twice with 10 mL prewarmed medium and incubating the dishes for 10 min at 4 • C. The supernatants were carefully removed and discarded, and the plates were washed once with prewarmed Dulbecco's phosphate-buffered saline solution (PBS) (Gibco Laboratories, Gaithersburg, MD, USA). Cold PBS (15 mL) containing 1.5% FBS was added, followed by the addition of 0.3 mL 0.1 M EDTA (pH 7.0). The viability of the detached cells was assessed by trypan blue exclusion, and the proportion of macrophages was determined after cytoplasmic staining with acridine orange using fluorescence microscopy. More than 95% of the cells were viable, and the cell preparations contained >95% macrophages.

Quantification of miRNA and mRNA Expression
For the quantitative analysis of miRNA expression, quantitative RT-PCR was performed using a TaqMan MicroRNA Reverse Transcription Kit, the TaqMan MicroRNA Assay (primer/probe sets for miRNAs), and TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA), according to the manufacturer's protocols. Relative miRNA expression was normalized to U6 small nuclear RNA. The results were analyzed using the 2 −∆∆ CT method.

Statistical Analyses
All data are presented as the means ± standard error of the mean (SEM) of three independent experiments. For comparisons between two groups, the Student's t-test was used. Multi-group comparisons of mean values were analyzed by a one-way analysis of variance followed by LSD post hoc test. Statistically significant differences were defined as p < 0.05.

PA Induces NLRP3 Inflammasome Activation in THP-1 Cells and Primary Murine Macrophages
To investigate the effect of PA on the transcription of NLRP3 inflammasome components, THP-1 cells were treated with 200 µM of PA-BSA conjugate for 2, 4, 8, 12, and 24 h. As shown in Figure 1A, the mRNA levels of NLRP3, caspase-1, IL-18, and IL-1β increased gradually in a time-dependent manner for 12 h at all concentrations. Western blotting was then performed to analyze the protein levels of these genes in the cell lysates and culture supernatants after THP-1 cells were treated with 200 µM PA for 4, 8, 12, and 24 h. The protein expression of the p10 fragment of mature caspase-1, IL-18, and IL-1β was enhanced in a similar manner as mRNA expression ( Figure 1B). Furthermore, significant levels of the p10 fragment of mature caspase-1, IL-18, and IL-1β were released into the culture supernatant ( Figure 1B). ELISA analysis revealed that PA enhanced caspase-1 activity and IL-1β secretion ( Figure 1C,D). In addition, we confirmed that PA induced NLRP3 inflammasome activation in primary murine macrophages, which resembled SFA-overloaded macrophages ( Figure 1E-H). These results suggest that PA induced NLRP3 inflammasome activation, which induced NLRP3 expression, caspase-1 activation, and caspase-1-dependent production of IL-18 and IL-1β in THP-1 cells and primary murine macrophages.

PA Up-Regulates miR-132 Expression in THP-1 Cells and Primary Murine Macrophages
We screened miRNAs up-regulated by PA treatment using microarray analyses. miR-132 expression was up-regulated >10-fold following PA treatment in THP-1 cells. We confirmed the effect of PA treatment on the expression of miR-132 using quantitative RT-PCR. The expression of miR-132 was increased in a time-dependent manner for 12 h when THP-1 cells were exposed to 200 µM PA (Figure 2A). Similarly, PA up-regulated miR-132 expression in primary murine macrophages ( Figure 2B).

miR-132 Regulates PA Induced-NLRP3 Inflammasome Activation in THP-1 Cells and Primary Murine Macrophages
To understand the role of miR-132 in NLRP3 inflammasome activation, THP-1 cells were transfected with a mimic negative control, miR-132 mimic, an inhibitor negative control, or miR-132 inhibitor, followed by stimulation with 200 µM PA for 12 h. Transfection of PA-untreated THP-1 cells with miR-132 mimic the significantly decreased mRNA expression of NLRP3, caspase-1, IL-18, and IL-1β compared to the mimic negative control ( Figure 3A). In addition, the miR-132 mimic significantly down-regulated the increased mRNA levels of NLRP3, caspase-1, IL-18, and IL-1β induced by PA treatment compared to the mimic negative control ( Figure 3A). Western blotting analysis of NLRP3 protein in cell lysates and supernatants revealed that the miR-132 mimic decreased the expression and release of the PA-induced NLRP3 inflammasome component ( Figure 3B). In contrast, as shown in Figure 3C-F, the miR-132 inhibitor enhanced the mRNA and protein expression of the NLRP3 inflammasome component, and increased caspase-1 activity and IL-1β secretion compared to the inhibitor negative control in THP-1 cells. Furthermore, inhibition of miR-132 increased PA-induced NLRP3 inflammasome activation in primary murine macrophages ( Figure 3G-I). These results imply that miR-132 is involved in PA-induced inflammasome activation, which can lead to caspase-1 activation as well as IL-1β and IL-18 processing and release.

miR-132 Does Not Regulate PA-Induced TLR Signaling in THP-1 Cells
To determine whether miR-132 regulates PA-induced TLR signaling, THP-1 cells were transfected with an inhibitor negative control or miR-132 inhibitor, followed by stimulation with 200 µM PA for 12 h. The mRNA levels of myeloid differentiation primary response 88 (MyD88), IL-1 receptor-associated kinase 1 (IRAK1), and TNF receptor-associated factor 6 (TRAF6) were examined. PA up-regulated the mRNA expression of MyD88, IRAK1, and TRAF6, and no significant differences in PA-induced MyD88, IRAK1, and TRAF6 mRNA expression were detected between inhibitor negative control and miR-132 inhibitor transfected cells ( Figure 4A). In addition, no significant difference in PA-induced NF-κB p65 protein expression was observed between inhibitor negative control and miR-132 inhibitor transfected cells ( Figure 4B). These data suggest that miR-132 does not regulate PA-induced TLR signaling in THP-1 cells.

FOXO3 Is a Target of miR-132
To elucidate the molecular functions of miR-132 in the regulation of PA-induced inflammasome activation, we screened predicted targets of these miRNAs in silico to identify genes related to inflammasome activation using TargetScan (www.targetscan.org) and microRNA (www.microrna.org); we identified FOXO3 ( Figure 5A). FOXO3 was recently validated as a direct target of miR-132 in neurons and bone marrow cells [18,19]. PA up-regulated FOXO3 mRNA and protein expression in THP-1 cells and primary murine macrophages ( Figure 5B,C,I,J). To investigate whether PA-induced FOXO3 activation is due to TLR-induced NF-κB activity, the mRNA levels of FOXO3 were examined in NF-κB-silenced cells. No significant difference in FOXO3 expression was detected between the siRNA control and NF-κB-silenced cells (Supplementary Materials Figure S1G).
To verify the miR-132 target FOXO3, we transfected a miR-132 mimic or miR-132 inhibitor into THP-1 cells and treated the cells with PA. The miR-132 mimic down-regulated FOXO3 mRNA and protein expression compared to the mimic negative control with or without PA treatment, while the miR-132 inhibitor up-regulated FOXO3 ( Figure 5D-G,K). No significant difference in PA-induced miR-132 expression was observed between the siRNA control and FOXO3 siRNA-transfected cells ( Figure 5H). Therefore, data from previous studies and our findings indicate that FOXO3 is a target of miR-132 in THP-1 cells.

The Silencing of FOXO3 Suppresses PA-Induced NLRP3 Inflammasome Activation in THP-1 Cells and Primary Murine Macrophages
To understand the direct influence of FOXO3 on PA-induced NLRP3 inflammasome activation, the mRNA and protein levels of NLRP3, caspase-1, IL-18, and IL-1β were examined in FOXO3-silenced cells. The transfection efficiency in 48 h post-transfected cells was confirmed by Western blot analysis ( Figure 6A). Compared to the scrambled siRNA control, FOXO3 siRNA-transfected cells exhibited a significant decrease in NLRP3, caspase-1, IL-18, and IL-1β gene expression in response to PA ( Figure 6B,F). In addition, Figure 6C shows that the protein levels of NLRP3, caspase-1, IL-18, and IL-1β in lysates and supernatants prepared from FOXO3-silenced cells decreased with or without PA treatment. The silencing of FOXO3 down-regulated PA-induced caspase-1 activity and IL-1β secretion ( Figure 6D,E,G,H). These results suggest that the suppression of FOXO3 inhibits the activation of NLRP3 inflammation.

NLRP3 Inflammasome Activation through miR-132 Inhibitor Is Attenuated by the Knockdown of FOXO3
We examined whether the knockdown of FOXO3 counteracts the NLRP3 inflammasome activation effect by inhibition of miR-132. Co-transfection with FOXO3 siRNA, but not control siRNA, significantly diminished the increase in mRNA and protein expression of the NLRP3 inflammasome component by miR132 inhibitor (Figure 7A,B). A similar pattern was observed in cells co-transfected with FOXO3 siRNA, showing decreased miR-132 inhibitor-induced caspase-1 activation and secretion of IL-1β ( Figure 7C,D). These findings indicate that miR-132 regulates NLRP3 inflammasome activation through down-regulation of FOXO3.

Schematic Diagram of the Proposed Model Showing How miR-132 Regulates PA-Induced NLRP3 Inflammasome Activation
On the basis of our findings, we suggest the model below ( Figure 8). PA induces the activation of the NLRP3 inflammasome and up-regulates FOXO3 and miR-132 expression. FOXO3, as a target of miR-132, is involved in PA-induced NLRP3 inflammasome activation. miR-132 serves as a negative feedback regulator in PA-induced NLRP3 inflammasome activation via FOXO3 down-regulation. On the basis of our findings, we suggest the model below ( Figure 8). PA induces the activation of the NLRP3 inflammasome and up-regulates FOXO3 and miR-132 expression. FOXO3, as a target of miR-132, is involved in PA-induced NLRP3 inflammasome activation. miR-132 serves as a negative feedback regulator in PA-induced NLRP3 inflammasome activation via FOXO3 down-regulation.

Discussion
Here, we report a potential mechanism of miR-132 in the regulation of PA-triggered NLRP3 inflammasome activation in THP cells and primary murine macrophages. Our results show that PA induced miR-132 expression as well as NLRP3 inflammasome activation. Overexpression of miR-132 was associated with a significant decrease in FOXO3 protein expression, along with a reduction in NLRP3, caspase-1 p10, IL-18, and IL-1β production in response to PA stimulation. Suppression of miR-132 resulted in the opposite results. In addition, silencing of FOXO3 suppressed PA-induced NLRP3 inflammasome activation. NLRP3 inflammasome activation through miR-132 inhibitor is attenuated by the knockdown of FOXO3. Collectively, these data suggest that miR-132 is an important modulator that plays a negative role in PA-induced NLRP3 inflammasome activation, at least in part through FOXO3.
Emerging evidence suggests that NLRP3 activation is involved in inflammatory responses, antimicrobial responses, and various human diseases, including autoimmune inflammatory disease, Alzheimer's disease, atherosclerosis, and type 2 diabetes [20]. Previous studies using genetically modified mice lacking NLRP3 inflammasome components showed that activation of the NLRP3 inflammasome is essential for the induction of obesity-induced inflammation and development of insulin resistance [9,10,21]. PA, one of the most abundant SFAs in the modern diet induces activation of the NLRP3 inflammasome in hematopoietic cells, which results in insulin resistance and glucose intolerance in mice [10]. In contrast, unsaturated fatty acids show a preventive effect on the activation of the NLRP3 inflammasome [22]. Furthermore, NLRP3 inflammasome activation is elevated in myeloid cells from type 2 diabetic patients [23].
The regulation of NLRP3 inflammasome-mediated IL-1β production in mononuclear phagocytes is cell-type specific [24]. In this study, untreated THP-1 cells expressed NLRP3 protein and released active caspase-1, IL-18, and IL-1β into the supernatant, and this was enhanced by PA treatment. These results suggest that the NLRP3 inflammasome is constitutively active in THP-1 cells. Therefore, primary signaling that induces the expression of pro-IL-1β mediated by the activation of TLRs through PA is sufficient for the production of IL-1β in THP-1 cells [22]. PA also potentiates the inflammasome by up-regulating the expression of NLRP3.
We found that miR-132 regulated PA-induced NLRP3 inflammasome activation, but not TLR signaling. Modulation of miR-132 had no effect on PA-induced MyD88, IRAK1, TRAF6, and NF-κB p65 expression, which are known to be important in TLR-4 and pro-inflammatory signaling.

Discussion
Here, we report a potential mechanism of miR-132 in the regulation of PA-triggered NLRP3 inflammasome activation in THP cells and primary murine macrophages. Our results show that PA induced miR-132 expression as well as NLRP3 inflammasome activation. Overexpression of miR-132 was associated with a significant decrease in FOXO3 protein expression, along with a reduction in NLRP3, caspase-1 p10, IL-18, and IL-1β production in response to PA stimulation. Suppression of miR-132 resulted in the opposite results. In addition, silencing of FOXO3 suppressed PA-induced NLRP3 inflammasome activation. NLRP3 inflammasome activation through miR-132 inhibitor is attenuated by the knockdown of FOXO3. Collectively, these data suggest that miR-132 is an important modulator that plays a negative role in PA-induced NLRP3 inflammasome activation, at least in part through FOXO3.
Emerging evidence suggests that NLRP3 activation is involved in inflammatory responses, antimicrobial responses, and various human diseases, including autoimmune inflammatory disease, Alzheimer's disease, atherosclerosis, and type 2 diabetes [20]. Previous studies using genetically modified mice lacking NLRP3 inflammasome components showed that activation of the NLRP3 inflammasome is essential for the induction of obesity-induced inflammation and development of insulin resistance [9,10,21]. PA, one of the most abundant SFAs in the modern diet induces activation of the NLRP3 inflammasome in hematopoietic cells, which results in insulin resistance and glucose intolerance in mice [10]. In contrast, unsaturated fatty acids show a preventive effect on the activation of the NLRP3 inflammasome [22]. Furthermore, NLRP3 inflammasome activation is elevated in myeloid cells from type 2 diabetic patients [23].
The regulation of NLRP3 inflammasome-mediated IL-1β production in mononuclear phagocytes is cell-type specific [24]. In this study, untreated THP-1 cells expressed NLRP3 protein and released active caspase-1, IL-18, and IL-1β into the supernatant, and this was enhanced by PA treatment. These results suggest that the NLRP3 inflammasome is constitutively active in THP-1 cells. Therefore, primary signaling that induces the expression of pro-IL-1β mediated by the activation of TLRs through PA is sufficient for the production of IL-1β in THP-1 cells [22]. PA also potentiates the inflammasome by up-regulating the expression of NLRP3.
We found that miR-132 regulated PA-induced NLRP3 inflammasome activation, but not TLR signaling. Modulation of miR-132 had no effect on PA-induced MyD88, IRAK1, TRAF6, and NF-κB p65 expression, which are known to be important in TLR-4 and pro-inflammatory signaling. Therefore, the regulatory effect of miR-132 on NLRP3 inflammasome activation may not be associated with TLR signaling as an upstream pathway.
As for FOXO3, the silencing of NF-κB had no effect on PA-induced FOXO3 expression. Although LPS can induce NLRP3 inflammasome activation and miR-132, it did not induce FOXO3 in THP-1 cells (Supplementary Figure S1A-F). These results suggest that PA-induced FOXO3 activation may not be the downstream effect of TLR-induced NF-κB activity. In addition, recent studies have demonstrated non-canonical activation of the inflammasome by LPS via caspase-4 and caspase-5, independent of the traditional LPS receptor TLR4 [25]. Thus, this may lead to differences in FOXO3 expression induced by PA and LPS. Further study is needed to clarify these differences.
The mechanism by which FOXO3 activates the NLRP3 inflammasome remains unknown. It has been reported that uncoupling protein 2 (UCP2), a known FOXO3 target, promotes NLRP3 inflammasome activation in macrophages [26]. Therefore, in an indirect pathway, it is possible that FOXO3 activates the NLRP3 inflammasome through UCP2. Additional studies on the direct action of FOXO3 on the NLRP3 inflammasome are necessary to confirm this indirect hypothesis.
Several studies have demonstrated the function of miR-132. miR-132 has also been implicated in neuronal differentiation and function [27,28]. miR-132 attenuates neuroinflammation by targeting acetylcholinesterase, and it can modulate inflammation induced by bacterial infection [14,29]. miR-132, which is induced by LPS or peptidoglycan, can act via negative feedback to prevent uncontrolled inflammation. In addition, miR-132 can regulate Kaposi's sarcoma-associated herpesvirus-induced inflammation [15]. Overexpression of miR-132 has been shown in chronic autoimmune disease conditions [30,31] and hematological malignancies [32]. Taken together, this suggests that miR-132 is an important regulator of the immune system.
To our knowledge, our study is the first to show the role of miR-132 in PA-induced NLRP3 inflammasome activation. In the current study, the mRNA expression levels of the NLRP3 inflammasome components, miR-132 expression, and FOXO3 mRNA expression were concurrently increased approximately 4 h after PA treatment. The protein expression levels of FOXO3 and the NLRP3 inflammasome components were increased 8 and 12 h after PA treatment, respectively. Furthermore, NLRP3 inflammasome activation through miR-132 inhibition was attenuated by FOXO3 knockdown. These findings suggest that miR-132 acts as a tuning regulator to prevent overactivation of the inflammasome by downregulating FOXO3.
Based on studies by Park et al. [33,34], even though Porphyromonas gingivalis induced IL-1β release via NLRP3 inflammasome activation and miR-132 expression, miR-132 did not affect inflammasome activation in THP-1 cells. The precise reasons for this difference are unclear, but it may be due to the different stimuli used.
The observed miR-132-mediated suppression of FOXO3 is similar to the findings of previous studies [18,19]. Until now, the relationship between miR-132 and FOXO3 has mostly been investigated in the context of cell survival because FOXO3 is a known aging-related pro-apoptotic transcription factor. Our results indicate that miR-132 suppresses inflammation activation through FOXO3.

Conclusions
In summary, our study identifies miR-132 as an important and novel negative feedback regulator of PA-induced NLRP3 inflammasome activation in THP-1 cells. Our results suggest that modulating miR-132 levels could be used in therapeutic interventions for inflammasome activation stimulated by high fat.

Conflicts of Interest:
The authors declare no conflict of interest.