MiR‐223‐3p inhibits rTp17‐induced inflammasome activation and pyroptosis by targeting NLRP3

Abstract The incidence of syphilis caused by Treponema pallidum subsp pallidum (T pallidum) infection is accompanied by inflammatory injuries of vascular endothelial cells. Studies have revealed that T pallidum infection could induce inflammasome activation and pyroptosis in macrophages. MicroRNA‐223‐3p (miR‐223‐3p) was reported to be a negative regulator in inflammatory diseases. The present study aimed to explore whether miR‐223‐3p regulates T pallidum‐induced inflammasome activation and pyroptosis in vascular endothelial cells, and determine the mechanisms which underlie this process. MiR‐223‐3p levels in syphilis and control samples were determined. The biological function of miR‐223‐3p in the NLRP3 inflammasome and pyroptosis was evaluated in T pallidum‐infected human umbilical vein endothelial cells (HUVECs). We observed a dramatic decrease in miR‐223‐3p levels in syphilis patients (n = 20) when compared to healthy controls (n = 20). Moreover, miR‐223‐3p showed a notable inhibitory effect on recombinant Tp17 (rTP17)‐induced caspase‐1 activation, resulting in decrease in IL‐1β production and pyroptosis, which was accompanied by the release of lactate dehydrogenase (LDH) in HUVECs. Additionally, the dual‐luciferase assay confirmed that NLRP3 is a direct target of miR‐223‐3p. Moreover, NLRP3 overexpression or knockdown largely blocked the effects of miR‐223‐3p on T pallidum‐induced inflammasome activation and pyroptosis in HUVECs. Most importantly, a notable negative correlation was observed between miR‐223‐3p and NLRP3, caspase‐1, and IL‐1β, respectively, in the serum of syphilis patients and healthy controls. Taken together, our results reveal that miR‐223‐3p targets NLRP3 to suppress inflammasome activation and pyroptosis in T pallidum‐infected endothelial cells, implying that miR‐223‐3p could be a potential target for syphilis patients.

negative regulator in inflammatory diseases. The present study aimed to explore whether miR-223-3p regulates T pallidum-induced inflammasome activation and pyroptosis in vascular endothelial cells, and determine the mechanisms which underlie this process. MiR-223-3p levels in syphilis and control samples were determined. The biological function of miR-223-3p in the NLRP3 inflammasome and pyroptosis was evaluated in T pallidum-infected human umbilical vein endothelial cells (HUVECs). We observed a dramatic decrease in miR-223-3p levels in syphilis patients (n = 20) when compared to healthy controls (n = 20). Moreover, miR-223-3p showed a notable inhibitory effect on recombinant Tp17 (rTP17)-induced caspase-1 activation, resulting in decrease in IL-1β production and pyroptosis, which was accompanied by the release of lactate dehydrogenase (LDH) in HUVECs. Additionally, the dual-luciferase assay confirmed that NLRP3 is a direct target of miR-223-3p. Moreover, NLRP3 overexpression or knockdown largely blocked the effects of miR-223-3p on T palliduminduced inflammasome activation and pyroptosis in HUVECs. Most importantly, a notable negative correlation was observed between miR-223-3p and NLRP3, caspase-1, and IL-1β, respectively, in the serum of syphilis patients and healthy controls.
Taken together, our results reveal that miR-223-3p targets NLRP3 to suppress inflammasome activation and pyroptosis in T pallidum-infected endothelial cells, implying that miR-223-3p could be a potential target for syphilis patients.

| INTRODUC TI ON
Syphilis is a multistage and chronic disease caused by Treponema pallidum subsp pallidum (T pallidum), which can involve multiple systems and is associated with significant morbidity. Globally, the incidence of syphilis infection is rising dramatically. 1,2 The inflammatory processes induced by syphilis within infected tissues result in the development of lesions. Moreover, the up-regulated expression or activation of NOD-like receptor (NLR), pyrin domain-containing (NLRP3) inflammasome, and the subsequent induction of pyroptosis result in excessive inflammation, which is responsible for the pathogenesis of various diseases. 3,4 Recently, studies have revealed that inflammasome responses are also involved in the development of syphilis-associated tissue inflammation. 5,6 Several factors, including bacteria, viruses, fungi, components of dying cells and crystal particles, can activate the NLRP3 inflammasome, which leads to caspase-1-dependent maturation of pro-interleukin(pro-IL)-1β and pro-IL-18. Eventually, NLRP3 inflammasome activation results in the induction of a cell death mechanism known as pyroptosis, via the cleavage of gasdermin D (GSDMD). [7][8][9] Recently, the outer membrane protein Tp92 of T pallidum was shown to have a potential pathogenic role by inducing a proinflammatory response in macrophages and human microvascular endothelial cells (HMEC)-1, 10 as well as inducing caspase-1-dependent pyroptosis of mononuclear cells. 11 Moreover, Li-Rong Lin et al revealed that T pallidum infection induced proinflammatory cytokine IL-1β secretion, which was accompanied by NLRP3 inflammasome activation, 6 suggesting the importance of the inflammasome and pyroptosis in T pallidum-induced pathology. Therefore, investigation of the factors that regulate NLRP3 inflammasome activation and pyroptosis may lead to the development of effective therapy strategies for syphilis patients.
MicroRNAs (miRNAs) are a highly conserved group of small non-protein-coding RNAs that act as post-transcriptional regulators of gene expression. Currently, there is increasing evidence, which demonstrates that miRNAs are involved in the regulation of many biological processes, including metabolism, inflammation and cancer. 12,13 Tp17 is a membrane immunogen of T pallidum, and our previous work has revealed that miR-216a-5p suppresses the recombinant Tp17 (rTP17)-induced inflammatory response in human umbilical vein endothelial cells (HUVECs) by targeting Toll-like receptor 4 (TLR4). 14,15 In addition, another study also reported that T pallidum membrane proteins may induce endothelial cell dysfunction and, consequently, affect the progress of the vessel inflammatory response. 10,16 Thus, we pondered whether there any other miRNAs involved in syphilis pathology. MiR-223-3p was previously reported to be up-regulated in tumour tissues and involved in regulating cell growth and apoptosis via the F-box/WD repeat-containing protein 7 (FBXW7), PR domain zinc finger protein 1 (PRDM1), Septin-6 (SEPT6), p53 and others. 17,18 In recent years, NLRP3 has also been reported to be a direct target of miR-223-3p. 19 However, the role and mechanism of action of miR-223-3p in syphilis remain unknown. Here, we aimed to investigate the effect of miR-223-3p in T pallidum-mediated inflammasome activation and pyroptosis in HUVECs. We have found that miR-223-3p induced a strong inhibition of T pallidum-induced caspase-1 activation and IL-1β production, which indicates that it might be a potential therapeutic target for syphilis.

| Cell cultures and materials
HUVECs were purchased from the BeNa Culture Collection and cultured in minimum essential medium (Invitrogen) supplemented with 10% foetal bovine serum (FBS), 100 U/mL penicillin and 0.1 mg/mL streptomycin. The cells, at a density of 10 4 viable cells/cm 2 , were seeded and then later used for experiments after 48 hours of culturing. The cells at passage 3 were used for this study, with three replicates for each condition.
The T pallidum Nichols strain was propagated in rabbits as previously described. 16

| Plasmids
The human NLRP3 coding region was cloned into a pCDNA3.0 vector. The 3ʹ-UTR of the NLRP3 mRNA containing the putative binding site of miR-223-3p was amplified and cloned into the

| ELISA assay
HUVECs, at a density of 5 ×

| Lactate dehydrogenase (LDH) assay
LDH release was quantified from the supernatants using the The percentage of LDH release was calculated using the following equation: LDH release (%) = (experimental LDH release -spontaneous LDH release)/maximum LDH release.
Protein concentrations were measured using the BCA protein assay

| Statistical analysis
Data are presented as mean ± standard deviation (SD) and 'n' represents the number of independent experiments. Statistical significance was calculated using Student's two-tailed unpaired t test or one-way analysis of variance (ANOVA) with Holm-Sidak's multiple comparisons test.
Moreover, correlation analysis was conducted using the Pearson correlation coefficient. P values of <.05 were considered statistically significant.
All statistical analyses were performed using the GraphPad Prism 8.0 software.

| Reduced miR-223-3p expression up-regulates caspase-1 and IL-1β in syphilis patients
To investigate the role of miR-223-3p in syphilis, we collected serum from 20 syphilis patients infected with T pallidum and 20 healthy controls. Similar to previous studies, caspase-1 and IL-1β mRNA ( Figure 1A-B) and protein ( Figure 1C-D) levels were found to be up-regulated in syphilis patients when compared with healthy controls. Notably, we found that miR-223-3p levels were strongly decreased in the serum of syphilis patients ( Figure 1E). Thus, we next evaluated the correlation between miR-223-3p expression and inflammasome activation. As shown in Figure 1F-G, miR-223-3p expression levels were found to be negatively correlated with caspase-1 and IL-1β levels, indicating that miR-223-3p may play a role in the progression of syphilis.

| Inflammasome activation and pyroptosis in rTp17-infected HUVECs
Studies on T pallidum membrane immunogens have provided insights into the immunopathogenesis of syphilis. Among the periplasmic lipoproteins, Tp17 has been reported to be a strong membrane immunogen and was initially recognized to play a role in inflammation during syphilis, as it reacted intensely with human syphilitic sera. 21,22 Moreover, our previous work also demonstrated that rTp17 could induce a strong inflammatory response in HUVECs (Peng et al, 2019). Therefore, in this study, we used the rTp17 as a stimulator and investigated whether the rTp17 could induce inflammasome activation and cell pyroptosis in HUVECs. As shown in Figure 2A-D, we found that treatment with 800 ng/mL of rTp17 for 24 hours was optimal for inducing inflammasome activation and pyroptosis, as well as up-regulated caspase-1 activation, IL-1β expression and LDH release. Thus, we used these conditions for all following experiments.

| Effect of miR-223-3p on rTp17-induced inflammasome activation and pyroptosis in HUVECs
To further determine the role of miR-223-3p in syphilis, we per-

| NLRP3 is the downstream target of miR-223-3p in HUVECs
To explore the mechanism by which miR-223-3p regulates inflammasome activation and cell pyroptosis in HUVECs, we investigated the downstream target genes associated with inflammasome activation. It was reported that NLRP3 transcription is tightly regulated by miR-223-3p through an evolutionarily conserved binding interaction between the 3ʹ-UTR of the NLRP3 mRNA and miR-223-3p in macrophages and hepatocellular carcinoma (HCC) cells. To confirm whether miR-223-3p could regulate NLRP3 expression in rTp17-treated HUVECs, we first constructed the 3ʹ-UTR of NLRP3 and inserted a point mutation into the site to prevent miR-223-3p binding ( Figure 4A). As shown in Figure 4B, co-transfection with miR-223-3p reduced the luciferase activity of HUVECs transfected with the NLRP3 containing the wild-type 3ʹ-UTR (NLRP3-3ʹ-UTR WT ) but not that of HUVECs transfected with the NLRP3 containing the mutated 3ʹ-UTR (NLRP3-3ʹ-UTR Mut ). Next, we further assessed the correlation between NLRP3 and miR-223-3p expression. Interestingly, NLRP3 expression in HUVECs was notably inhibited at the mRNA and protein F I G U R E 2 Inflammasome activation and pyroptosis occur in rTP17-infected HUVECs. HUVECs were treated with rTp17 at various concentrations (400 ng/mL, 800 ng/mL, 1500 ng/mL) and for various time-points (6, 12, 24 h). A, IL-1β levels in the cell supernatants were analysed with ELISA. B, The activation of caspase-1 in HUVECs was analysed with immunoblot using the anti-Casp-1 p10 antibody. C, LDH release was measured using an LDH release assay and the spontaneous release of LDH from HUVECs in the absence of rTp17 treatment was used as a control. The percentage of LDH release was calculated using the following equation: LDH release (%) = (Experimental LDH release -Spontaneous LDH release) / Maximum LDH release. D, miR-223-3p levels in HUVECs exposed to rTp17. *P < .01, **P < .01, and ***P < .001 vs the 6-h treatment group, Student's t test levels following treatment with a miR-223-3p mimic in either the presence or absence of an rTp17 infection. In contrast, treatment with the miR-223-3p inhibitor was shown to up-regulate NLRP3 expression ( Figure 4C-D). Moreover, an increase in NLRP3 expression was also observed in syphilis patients when compared to healthy control, which was negatively correlated with the expression of miR-223-3p ( Figure 4E-F). These data suggest that miR-223-3p could target NLRP3 in order to down-regulate its expression in HUVECs infected with rTp17.

| NLRP3 rescues the effect of miR-223-3p on rTp17-induced inflammasome activation and cell pyroptosis
To further confirm that the miR-223-3p-mediated inhibitory effect on inflammasome activation and cell pyroptosis was dependent on its target NLRP3, we constructed an NLRP3 plasmid to perform the rescue experiment. The immunoblot results showed that miR-223-3p-mediated down-regulation of NLRP3 was completely res-

| D ISCUSS I ON
In this study, we first demonstrated that miR-223-3p expression levels were decreased in syphilis patients when compared to healthy control, which was negatively correlated with caspase-1 activation and IL-1β expression. Furthermore, we found that treatment with F I G U R E 3 Effect of miR-223-3p on rTp17-induced inflammasome activation and pyroptosis in HUVECs. HUVECs were transfected with a mimic negative control (NC), miR-223-3p mimic, inhibitor NC, or miR-223-3p inhibitor for 48 h and then the cells were treated with rTp17 at 800 ng/mL for 12 h. A, The transfection efficiency for the miR-223-3p mimic or inhibitor was analysed with qRT-PCR. B, The activation of caspase-1 in HUVECs was analysed with immunoblotting using the anti-Casp-1 p10 antibody. C, The protein expression of IL-1β in HUVECs was analysed with Western blotting. D, LDH release was measured using an LDH release assay and the spontaneous release of LDH from untransfected HUVECs was used as a control. The percentage of LDH release was calculated using the following equation: LDH release (%) = (experimental LDH release -spontaneous LDH release) / maximum LDH release. **P <.01 and ***P < .001 vs mimic NC or inhibitor NC or control, Student's t test a miR-223-3p mimic strongly suppressed T pallidum-induced caspase-1 activation and IL-1β production, as well as LDH release, by directly targeting NLRP3. Conversely, treatment with a miR-223-3p inhibitor was found to have the opposite effect. Notably, we showed that NLRP3 overexpression could block the effect of miR-223-3p on T pallidum-induced inflammasome activation and pyroptosis. Thus, our study provides novel insights with regard to the role of miR-223-3p in T pallidum-mediated inflammasome activation and pyroptosis and suggests that miR-223-3p might be a promising therapeutic target for syphilis.
Syphilis is a chronic infectious disease caused by the spirochaete T pallidum and is usually venereal in origin but often congenital. Accumulating evidence has shown that T pallidum infection-induced tissue inflammation and injury, including inflammasome activation, contributes to the development of syphilis. 23,24 Moreover, a recent work revealed that the outer membrane protein Tp92 of T pallidum could induce caspase-1-dependent pyroptosis in HUVECs. 11 In addition, our previous study also demonstrated that Tp17, which is a strong membrane immunogen and reacts intensely with human syphilitic sera, induced TLR4 pathway activation and a proinflammatory response in HUVECs. 14 However, whether Tp17 could induce inflammasome activation and caspase-1-dependent pyroptosis remained unclear.
Here, we found that rTP17 stimulation could lead to caspase-1 activation and LDH release in the HUVECs.
Currently, miRNAs have emerged as an important therapeutic target for many diseases, including metabolic diseases, inflammation and cancer. MiR-223-3p was found to be down-regulated in F I G U R E 4 NLRP3 is the downstream target of miR-223-3p in HUVECs. A, Alignment of miR-223-3p and its corresponding complementary binding sequence in the 3ʹ-UTR of NLRP3 using the TargetScan bioinformatics algorithm. B, HUVECs were transfected with luciferase reporter plasmids containing the wild-type (WT) or mutant (Mut) NLRP3 3ʹ-UTR or an empty vector (Vector) together with a miR-223-3p mimic or mimic negative control (NC) and luciferase activity was measured at 48 h after transfection. C-D, HUVECs were transfected with a mimic NC, miR-223-3p mimic, inhibitor NC or miR-223-3p inhibitor for 48 h, and then, the cells were treated with or without rTp17 at 800 ng/mL for 12 h. NLRP3 expression was analysed with qRT-PCR and Western blotting. E, NLRP3 expression was analysed with qRT-PCR in blood samples from syphilis patients infected with T. pallidum and healthy controls (n=20 biological replicates). F, Correlation analysis of miR-223-3p and NLRP3 expression using Pearson's correlation coefficient. **P < .01 and ***P < .001 vs healthy controls, Student's t test several tumour types and is known to be involved in regulating cell growth and apoptosis. 25 Recently, several reports have confirmed that miR-223 directly targets NLRP3. [32][33][34] Our results also confirmed that miR-223-3p negatively regulates the NLRP3 expression, by using a dual-luciferase assay, and also that this process occurs in syphilis patients and HUVECs stimulated with rTP17. Moreover, we found that NLRP3 overexpression could block the inhibitory effects of miR-223-3p on inflammasome activation and pyroptosis in the HUVECs stimulated with rTP17.
In conclusion, we found that miR-223-3p was significantly down-regulated in syphilis patients when compared to healthy controls, whereas NLRP3 and caspase-1 expression were dramatically up-regulated in syphilis patients. Moreover, miR-223-3p inhibited HUVECs transfected with an empty vector or an NLRP3 plasmid were further transfected with or without a mimic negative control (NC) or miR-223-3p mimic for 48 h. A, The expression of NLRP3 in HUVECs was analysed with Western blotting. The transfected cells were then treated with rTp17 at 800 ng/mL for 12 h. B, The activation of caspase-1 in HUVECs was analysed with immunoblotting using an anti-Casp-1 p10 antibody. C, The protein expression of IL-1β in HUVECs was analysed with Western blotting. D, LDH release was measured using an LDH release assay and the spontaneous release of LDH from HUVECs transfected with an empty vector + mimic NC was used as a control. The percentage of LDH release was calculated using the following equation: LDH release (%) = (experimental LDH release -spontaneous LDH release) /maximum LDH release. **P < .01 and ***P < .001 vs mimic NC or inhibitor NC or control, Student's t test to our understanding of syphilis pathogenesis and also supplies a potential therapeutic target for syphilis.

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.

F I G U R E 6
Effect of NLRP3 knockdown on the miR-223-3p-mediated regulation of inflammasome activation and cell pyroptosis. HUVECs transfected with a negative control (NC) or NLRP3 siRNA were further transfected with or without an inhibitor NC or miR-223-3p inhibitor for 48 h. A, The expression of NLRP3 in HUVECs was analysed with Western blotting. The transfected cells were then further treated with rTp17 at 800 ng/mL for 12 h. B, The activation of caspase-1 in HUVECs was analysed with immunoblotting using an anti-Casp-1 p10 antibody. C, The protein expression of IL-1β in HUVECs was analysed with Western blotting. D, LDH release was measured. The percentage of LDH release was calculated using the following equation: LDH release (%) = (experimental LDH release -spontaneous LDH release) / maximum LDH release. **P < .01 and ***P < .001 vs mimic NC or inhibitor NC or control, Student's t test