The inflammatory experiments and infection assays were conducted on the human monocyte/macrophage-like cell line THP-1 (ATCC: TIB-202™, ATCC, Manassas, VA, USA) while the HEK 293T (Human Embryonic Kidney) cells (ATCC: CRL-3216™, ATCC, Manassas, VA, USA) were used for in vitro validation. Vero epithelial cells (ATCC: CCL-81™) were utilized for HSV-2 propagation (ATCC: VR-734D™, ATCC, Manassas, VA, USA). HEK 293T and Vero cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Waltham, MA, USA), while THP-1 cells were cultured in RPMI-1640 medium (Gibco, Waltham, MA, USA). All media were supplemented with 10% fetal bovine serum (FBS) (Invitrogen, Waltham, MA, USA), 20 mM HEPES, and 1% penicillin-streptomycin. Additionally, THP-1 cells were supplemented with 1 mM sodium pyruvate. THP-1 cells were differentiated into macrophages using 10 nM Phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich, St. Louis, MO, USA) for 24 hours, followed by a 24-hour rest period in PMA-free media. All cell cultures were maintained at 37°C in a 5% CO₂ humidified atmosphere, regularly monitored for mycoplasma contamination, and handled according to ATCC guidelines.

HSV-2 (ATCC: VR-734D™, ATCC, Manassas, VA, USA) was propagated in Vero cells under serum-starved conditions with 2% FBS for 2–3 days. Viral titers were quantified using a standard plaque assay technique (Huleihel et al., 2001).

HSV-2 infection was given at MOI 1 while LPS treatment was administered at the concentration of 10μg/ml for 24 hours. Total RNA was extracted using the TRIzol (Invitrogen, Waltham, MA, USA) organic extraction method, and cDNA was synthesized via reverse transcription using the SuperScript™ III First-Strand Synthesis System (Invitrogen: 18080-051). Infection was confirmed by amplifying the HSV-2 viral gene UL30 using Power SYBR™ Green PCR Master Mix (Applied Biosystems™, Madison, WI, USA: 4367659) with 200 nM UL30-specific primers. The reduction in viral mRNA levels (UL30, RL2, and UL44) was assessed post-transfection with miRNA mimics. All qPCR experiments adhered to MIQE guidelines, with GAPDH as the internal reference for normalization. Relative changes in mRNA levels were calculated using the 2^−ΔΔCt method, and results were expressed as fold changes relative to controls. Primer sequences are listed in Supplementary Table S1 .

Cell supernatants were collected at designated time points to measure IL-1β levels using the Human IL-1β ELISA Kit (BMS224-2, Invitrogen, Waltham, MA, USA) and IL-18 levels using the Human IL-18 ELISA Kit (DBP180, R&D Systems, Minneapolis, MN, USA) as per the manufacturers’ instructions. Briefly, samples were applied to ELISA plates pre-coated with IL-1β- or IL-18- specific antibodies, followed by detection antibodies and substrates. Absorbance was measured at the specified wavelength to quantify cytokine levels.

The miRNAs hsa-miR-141-3p and hsa-miR-211-5p were selected for this study based on preliminary screening using the Human Inflammatory and Immune Response miRNA PCR Array (Qiagen, Cat. No. MIHS-105ZA), which revealed their downregulation in HSV-2-infected THP-1 cells (Banerjee et al., 2023). These miRNAs are the mature, functional strands derived from the 3′ arm of pre-miR-141 and the 5′ arm of pre-miR-211, respectively, as annotated in miRBase (release 22.1). To identify putative targets relevant to inflammasome activation, we performed in silico predictions using three independent databases: miRWalk (http://mirwalk.umm.uni-heidelberg.de/ accessed on July 12, 2022), TargetScanHuman 8.0 (https://www.targetscan.org/vert_80/ accessed on January 25, 2023), miRDB (http://mirdb.org/ accessed on January 29, 2023) (Agarwal et al., 2015; Sticht et al., 2018; Chen and Wang, 2020). These analyses consistently predicted NLRP3 as a target of hsa-miR-141-3p and CASP1 as a target of hsa-miR-211-5p. The seed region complementarity and binding interactions for these targets were visualized based on TargetScanHuman 8.0 output. The combination of predictions from multiple platforms strengthened the confidence in selecting these target genes for experimental validation.

To validate miRNAs involved in the inflammatory pathway, TaqMan-based quantitative PCR was employed. miRNAs from HSV-2-infected THP-1 cells were extracted using the mirVana™ miRNA Isolation Kit (Thermo Fisher Scientific, Cat. No. AM1560). Specific miRNAs were validated using TaqMan primer-probe assays for hsa-miR-141-3p (Assay ID: 000463) and hsa-miR-211-5p (Assay ID: 00051674). Reverse transcription and real-time PCR were performed using the TaqMan™ MicroRNA Reverse Transcription Kit, optimized for low RNA input. RNA-U6 was used as the endogenous control for normalization. Relative miRNA expression was calculated using the 2^−ΔΔCt method, with graphical representation highlighting miRNA expression dynamics.

The 3′ UTR regions of NLRP3 and CASP1 genes, involved in inflammasome assembly and predicted as targets of miR-141 and miR-211, were amplified using gene-specific primers ( Supplementary Table S2 ) with MluI and HindIII restriction sites. PCR amplicons and pMIR-REPORT luciferase vector (Ambion, Austin, TX, USA: AM5795) were digested with MluI and HindIII (New England Biolabs, Rowley, MA, USA: R0198S and R0104S) and ligated using T4 DNA Ligase (New England Biolabs, M0202S). Ligation products were transformed into DH5α competent cells, and recombinant plasmids were extracted using the QIAprep Spin Miniprep Kit (Qiagen, Hilden, Germany: 27106).

The recombinant plasmids containing the cloned 3′ UTR regions were then transfected or co-transfected into HEK293T or THP-1 cells using Lipofectamine 2000 (Invitrogen, Waltham, MA, USA: 11668019) following the manufacturer’s instruction. Functional studies utilized miRNA mimics (50 nM) for miR-141-3p (MC10860) and miR-211-5p (MC10168), along with scrambled miR control obtained from Thermo Fisher Scientific (Waltham, MA, USA).

To validate miRNA targets, wild-type and mutated 3′ UTRs of NLRP3 and CASP1 were cloned into pMIR-REPORT plasmids and transfected into HEK293T cells along with miRNA mimics or scrambled miR controls. Luciferase activity was measured using the Luciferase Reporter Gene Detection Kit (Sigma-Aldrich, LUC1-1KIT). Reduced luciferase activity confirmed miRNA binding to target genes.

Cells were harvested, and lysates were prepared using a lysis buffer supplemented with protease inhibitors to maintain protein integrity. The lysates were subjected to protein separation via electrophoresis on a 10% polyacrylamide gel, followed by transfer onto a PVDF membrane. The membrane was blocked with 5% non-fat dried milk for 1 hour at room temperature and incubated with primary antibodies specific to NLRP3 (15101), ASC (13833), Cleaved CASP1 (p20 subunit - Asp297; 4199), IL-1β (12703), IL-18 (57058), Cleaved GSDMD (Asp275; 36425) procured from Cell Signaling Technology (Danvers, MA, USA), HSV-2 gB (57857; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and HSV-2 ICP8 (56992; Santa Cruz Biotechnology, Santa Cruz, CA, USA). The proteins bound to the primary antibodies were detected using either horseradish peroxidase (HRP)-conjugated primary or secondary antibodies. Signals were visualized using Pierce™ ECL Western Blotting Substrate (Thermo Scientific, Waltham, MA, USA), and chemiluminescence was captured using a ChemiDoc imaging system (Bio-Rad, Hercules, CA, USA). The membrane was stripped using 1× ReBlot Plus Strong Antibody Stripping Solution (Merck-Millipore, Burlington, MA, USA: 2504), re-blocked, and re-probed with a GAPDH antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA: 47724-HRP) as an internal loading control. Protein band intensities were quantified using ImageJ software (NIH, version 1.53a) and normalized to GAPDH levels for comparative analysis.

Statistical significance was determined using GraphPad Prism v9.0. Data were analyzed using one-way ANOVA or Student’s t-test, and results were presented as mean ± standard deviation (SD) from at least three independent experiments. P-values < 0.05 were considered statistically significant.

Upon viral infection, pattern recognition receptors (PRRs) sense viral entry and trigger inflammatory responses (Li and Wu, 2021). While HSV-1 is known to upregulate NLRP3 inflammasome activation, the mechanism by which HSV-2 mediates inflammation remains unclear (Li et al., 2019; Vitali et al., 2020). Given that NLRP3 is the most studied inflammasome in viral infections (Zheng et al., 2023), we investigated whether HSV-2 triggers inflammation through NLRP3 inflammasome activation in THP-1-derived macrophages.

To determine the expression of key inflammasome components, we performed quantitative PCR for NLRP3, ASC, and CASP1 – the essential inflammasome components, along with UL30 to confirm HSV-2 infection. THP-1 cells were infected with HSV-2 at 1 MOI, and samples were collected at 4-, 8-, 16-, and 24-hours post-infection (hpi). LPS-treated cells served as a positive control. A significant increase in NLRP3 mRNA expression was observed with progressing infection, peaking at 16 hpi (11-fold increase compared to mock-infected cells) ( Figure 1A ). ASC mRNA expression also increased significantly at 16 hpi (23-fold increase), followed by declining at 24 hpi while remaining elevated compared to earlier time points ( Figure 1B ). CASP1 mRNA levels showed a dramatic upregulation, peaking at 16 hpi (170-fold increase) and sustaining at 24 hpi ( Figure 1C ). Meanwhile, UL30 mRNA levels increased significantly at 16 hpi ( Figure 1D ). The inflammatory receptor NLRP3 and its downstream components, ASC and CASP1, exhibited peak mRNA expression at 16 hpi, which then declined by 24 hpi ( Figures 1A-C ), suggesting temporally regulated transcriptional and translational dynamics.

To validate these findings at the protein level, we performed Western blot analysis for NLRP3, ASC, Cleaved CASP1, HSV-2 ICP8, and GAPDH at different time points as indicated ( Figure 1E ). Compared to mock-infected cells, HSV-2 infection led to an increase in NLRP3, ASC, and cleaved CASP1 protein levels, with quantification confirming peak expression at 24 hpi ( Supplementary Figure S1 ), confirming NLRP3 inflammasome activation upon HSV-2 infection. LPS-treated cells exhibited robust activation of inflammasome markers, confirming the validity of the experimental setup. HSV-2 infection at 24 hpi led to similar protein expression of the inflammatory markers as the 24 hours-LPS-treated cells ( Figure 1F ; Supplementary Figure S2 ). Additionally, the HSV-2 ICP8 protein, a marker of viral replication, showed a consistent increase over time, peaking at 24 hpi, confirming viral progression. These findings confirm that mRNA expression of inflammatory markers peaks at 16 hpi, followed by significant protein expression at 24 hpi, reflecting a transition from transcriptional activation to translational regulation. Overall, these results suggest that HSV-2 infection triggers NLRP3 inflammasome activation, leading to increased inflammatory responses.

The activation of inflammasome plays an important role in the innate immune response to infections, including their involvement in pyroptosis, a pro-inflammatory form of programmed cell death (Guo et al., 2015). Inflammasome activation often leads to the cleavage of Gasdermin-D (GSDM-D) and subsequent secretion of inflammatory cytokines, such as IL-1β and IL-18, which amplify inflammatory responses (Yao et al., 2024). HSV-1 has been shown to induce pyroptosis, thereby exacerbating inflammation (Hu et al., 2022; Jiang et al., 2024). Similarly, HSV-2 has also been shown to induce cell death, which may further contribute to inflammation (Vitali et al., 2020; Dass et al., 2024). Yet the mechanisms driving HSV-2-induced pyroptosis remain unexplored. To investigate whether HSV-2 infection induces pyroptosis, we analyzed the expression of IL-1β, IL-18, and GSDM-D at the mRNA and protein levels in HSV-2-infected THP-1 cells.

qPCR analysis revealed a significant increase in IL-1β, IL-18, and GSDM-D mRNA expression upon HSV-2 infection ( Figures 2A-C ). IL-1β levels increased steadily, reaching a 200-fold change at 16 hpi ( Figure 2A ), while IL-18 expression peaked at 16 hpi (4-fold increase) before declining at 24 hpi ( Figure 2B ). Similarly, GSDM-D mRNA levels exhibited peak upregulation at 16 hpi (25-fold increase), suggesting activation of the pyroptotic pathway ( Figure 2C ). UL30 expression continued to increase, confirming active HSV-2 replication ( Figure 2D ).

To confirm these results, we performed Western blot analysis for IL-1β, IL-18, and GSDM-D in infected THP-1 cells ( Figure 2E ). Protein expression of mature IL-1β and IL-18 progressively increased over time ( Supplementary Figure S3 ). Additionally, the cleavage of GSDM-D, a hallmark of pyroptotic cell death, was detected, with levels increasing upon HSV-2 infection ( Supplementary Figure S3 ). Furthermore, analysis of culture supernatants from HSV-2-infected THP-1 cells revealed a marked increase in IL-1β and IL-18 secretion, peaking at approximately 1200 pg/mL and 550 pg/mL, respectively, at 24 hpi ( Figures 2F-G ).

Collectively, these findings demonstrate that HSV-2-induced inflammasome activation leads to pyroptosis, as indicated by increased IL-1β and IL-18 expression and release, along with the proteolytic cleavage of GSDM-D. These data highlight pyroptosis as a key component of HSV-2 pathogenesis.

miRNAs serve as key regulators of inflammation and immune responses, and recent studies suggest their involvement in HSV-2 pathogenesis (Wang et al., 2022; Banerjee et al., 2023; Yan et al., 2023; Long et al., 2024; Zhan et al., 2024). Among various miRNAs associated with immune modulation, miR-141 and miR-211 were selected based on preliminary array screening of HSV-2-infected macrophages, which revealed significant downregulation of these miRNAs (Banerjee et al., 2023). Both are predicted to target core components of the NLRP3 inflammasome pathway (NLRP3 and CASP1, respectively), and prior studies have reported their roles in suppressing inflammatory responses and pyroptosis (Wang et al., 2022; Yan et al., 2023; Long et al., 2024; Zhan et al., 2024). Given the strong association between HSV-2 infection and excessive inflammation, we sought to determine whether HSV-2 modulates miR-141 and miR-211 expression, potentially contributing to viral pathogenesis.

To assess the expression dynamics of these miRNAs during HSV-2 infection, we performed TaqMan-based qPCR on total RNA extracted from HSV-2-infected THP-1 macrophages at 8-, 16-, and 24 hpi. Our qPCR analysis demonstrated a significant downregulation of both miRNAs miR-141 and miR-211 upon HSV-2 infection, with distinct temporal patterns ( Figures 3A, B ). At 8 hpi, miR-141 expression was markedly reduced to 0.40-fold compared to mock-infected controls, indicating a substantial suppression of miR-141 during the early phase of infection ( Figure 3A ). Conversely, miR-211 expression showed no significant change at this time point ( Figure 3B ). At 16 hpi, both miRNAs were significantly downregulated. By 24 hpi, miR-141 expression showed partial recovery ( Figure 3A ), whereas miR-211 levels exhibited a more pronounced decline, reaching 0.48-fold of mock-infected controls ( Figure 3B ), suggesting enhanced suppression during the later stage of infection. Notably, LPS treatment did not significantly alter miR-141 or miR-211 expression levels ( Figures 3A, B ), indicating that their downregulation is specific to HSV-2 infection rather than a general inflammatory response. These findings suggest that miR-141 downregulation occurs predominantly during early HSV-2 infection, whereas miR-211 suppression intensifies at later stages, possibly reflecting differential miRNA regulation as viral replication progresses. UL30 mRNA expression, a viral gene encoding the HSV-2 DNA polymerase catalytic subunit, was used as an indicator of viral replication kinetics across these time points ( Figure 3C ).

Given the significant downregulation of miR-141 and miR-211 during HSV-2 infection, we sought to determine whether these miRNAs regulate key inflammatory pathways associated with HSV-2-induced immune responses. Using in silico analysis, we employed the miRWalk database (http://mirwalk.umm.uni-heidelberg.de/, accessed on July 12, 2022) to predict potential target genes of miR-141 and miR-211 that may be involved in inflammasome activation. Our analysis identified NLRP3 and CASP1 as putative targets of miR-141 and miR-211, respectively. These genes encode key components of the NLRP3 inflammasome, a critical regulator of inflammatory responses leading to IL-1β and IL-18 maturation and pyroptosis.

To validate these computational predictions, we performed a luciferase reporter assay using wild-type and mutated 3′ UTR sequences of NLRP3 and CASP1 cloned into the pMIR-REPORT luciferase vector. HEK293T cells were co-transfected with either miR-141 or miR-211 mimics, along with the corresponding wild-type or mutated 3′ UTRs of NLRP3 or CASP1. Scrambled miRNA controls (sc-miR) were used as negative controls to account for non-specific effects. Our results demonstrated a significant suppression of luciferase activity upon co-transfection with wild-type NLRP3 and miR-141 mimics, indicating a direct interaction between miR-141 and NLRP3 ( Figure 4A ). Specifically, luciferase activity was reduced by 74-80% in co-transfected cells containing the wild-type NLRP3 3′ UTR, whereas no significant reduction was observed in co-transfected cells containing the mutated NLRP3 3′ UTR, confirming the specificity of miR-141–NLRP3 interaction ( Figure 4A ). Similarly, co-transfection of miR-211 mimics with wild-type CASP1 3′ UTR resulted in a 50-54% decrease in luciferase activity compared to control cells, whereas the mutated CASP1 3′ UTR exhibited no significant change in luciferase activity ( Figure 4B ). These findings validate that miR-141 directly targets NLRP3, while miR-211 targets CASP1, establishing their functional roles in regulating inflammasome activation ( Figures 4A, B ). To further confirm the regulatory effects of miR-141 and miR-211, we assessed NLRP3 and CASP1 protein levels in THP-1 macrophages transfected with miRNA mimics or scrambled controls. Western blot analysis revealed that transfection with miR-141 mimic led to a substantial decrease in NLRP3 protein expression ( Figure 4C – left panel), whereas miR-211 mimic transfection resulted in a notable reduction in CASP1 protein levels ( Figure 4C – right panel). Scrambled miRNA controls had no effect on NLRP3 or CASP1 expression, further supporting the specificity of miR-141 and miR-211 in regulating inflammasome components.

Taken together, these results establish miR-141 and miR-211 as key post-transcriptional regulators of inflammasome activation, directly targeting NLRP3 and CASP1, respectively. The suppression of these miRNAs upon HSV-2 infection likely contributes to enhanced inflammasome activation, increased IL-1β and IL-18 secretion, and exacerbated inflammation. These findings indicate that miR-141 and miR-211 act as negative regulators of HSV-2-induced inflammatory responses and suggest their potential as therapeutic targets for controlling excessive immune activation during HSV-2 infection.

Having established miR-141 and miR-211 as key regulators of NLRP3 and CASP1, we next investigated whether their ectopic expression could mitigate HSV-2 replication. THP-1-derived macrophages were transfected with either miR-141 or miR-211 mimics (50 nM) or a scrambled miRNA (sc-miR) control prior to infection with HSV-2 (MOI = 1). At 8 hpi, the expression levels of key HSV-2 viral genes (RL2, UL30, and UL44) were quantified by qPCR ( Figure 5 ).

Quantitative PCR analysis revealed a significant reduction in viral gene expression upon miR-141 or miR-211 mimic transfection. In miR-141-transfected macrophages, RL2 expression was reduced by 89% compared to sc-miR-transfected cells ( Figure 5A ), while miR-211-transfected macrophages showed a 98.6% reduction ( Figure 5B ). Similarly, UL30 expression was suppressed by 97.4% and 70.5% following miR-141 and miR-211 mimic transfection, respectively ( Figures 5C, D ). UL44 expression was also substantially reduced by 57.5% and 78.8% in miR-141- and miR-211-transfected cells, respectively ( Figures 5E, F ). To assess the impact on infectious virion production, culture supernatants from miRNA mimic-transfected, HSV-2-infected THP-1 cells were analyzed by plaque assay. A 70–85% reduction in infectious virus titers was observed compared to sc-miR controls ( Supplementary Figure S4 ). These findings demonstrate that miR-141 and miR-211 interfere with multiple stages of the HSV-2 replication cycle, highlighting their potential as antiviral therapeutic candidates.

Since NLRP3 and CASP1 are primary targets of miR-141 and miR-211, respectively, we next examined whether ectopic miRNA expression could modulate inflammasome activation during HSV-2 infection. THP-1-derived macrophages transfected with miR-141 or miR-211 mimics were infected with HSV-2 (MOI = 1), and expression levels of NLRP3, GSDM-D, IL-1β, and IL-18 were quantified by qPCR at 8 hpi ( Figure 6 ).

Transfection with miR-141 mimics resulted in a significant downregulation of NLRP3 expression (78.6%), IL-1β (92.5%), IL-18 (73.2%), and GSDM-D (84.6%) compared to infected controls ( Figures 6A–D ). Similarly, miR-211 mimic transfection suppressed NLRP3 by 63.0%, IL-1β by 87.5%, IL-18 by 76.5%, and GSDM-D by 58.9% ( Figures 6E–H ).

To further confirm the proviral role of inflammasome activation in HSV-2 replication, THP-1 macrophages were pretreated with MCC950 (NLRP3 inhibitor) or Z-VAD-FMK (pan-caspase inhibitor) before infection. Both inhibitors significantly reduced HSV-2 UL30 and UL44 gene expression ( Supplementary Figure S5 ) and decreased viral protein levels of glycoprotein B (gB) and ICP8, as shown by Western blot analysis ( Supplementary Figure S6 ).

Additionally, Western blot analysis of HSV-2-infected cells transfected with miR-141 or miR-211 mimics demonstrated reduced expression of cleaved CASP1, cleaved IL-1β, IL-18, and GSDM-D proteins compared to control-transfected cells ( Figures 7A, B ). ELISA quantification of secreted IL-1β and IL-18 also revealed a significant decrease (63–66% for IL-1β and 73–84% for IL-18) in miRNA mimic-transfected cells relative to controls ( Figures 7C, D ).

Collectively, these findings demonstrate that miR-141 and miR-211 not only suppress HSV-2 replication but also mitigate inflammasome activation and pyroptotic inflammation by targeting key components of the NLRP3 inflammasome pathway. These results highlight the therapeutic potential of miRNA-based strategies in controlling HSV-2-induced immunopathology and viral dissemination.