MicroRNAs as Regulators of NLRP3 Inflammasome Activation in Herpes Simplex Virus Type 2 Infection

Debashree Dass¹Anwesha Banerjee¹Ashwini More¹Anupam Mukherjee^1,2*

• ¹ICMR-National Institute of Translational Virology and AIDS Research, Pune, India
• ²Academy of Scientific and Innovative Research (AcSIR), New Delhi, National Capital Territory of Delhi, India

Introduction: Herpes Simplex Virus Type 2 is a prevalent sexually transmitted pathogen that causes genital herpes and severe neurological complications, including meningitis and encephalitis. A major challenge in HSV-2 infection is the uncontrolled inflammatory response mediated by NLRP3 inflammasome activation, leading to pyroptosis and excessive cytokine secretion. Despite its significant clinical burden, the molecular mechanisms underlying HSV-2-induced inflammation remain poorly understood. Recent evidence suggests that microRNAs play a crucial role in regulating host immune responses and inflammasome activation. In this study, we investigate the regulatory role of miR-141 and miR-211 in modulating inflammasome activation and viral replication during HSV-2 infection.Methods: THP-1-derived macrophages were transfected with miR-141 or miR-211 mimics or scrambled controls before infection with HSV-2. Quantitative PCR and Western blot analysis were performed to assess the expression of NLRP3, CASP1, IL-1β, IL-18, and GSDM-D. Luciferase reporter assays were conducted to validate miRNA–target interactions, and ELISA was used to quantify cytokine levels in culture supernatants.Results: Our results demonstrate that HSV-2 infection significantly downregulates miR-141 and miR-211, leading to enhanced NLRP3 inflammasome activation, increased caspase-1 cleavage, and excessive secretion of IL-1β and IL-18, ultimately causing pyroptotic cell death. Transfection with miR-141 and miR-211 mimics restored miRNA expression, resulting in a marked suppression of inflammasome activation and inflammatory cytokine release, as well as significant inhibition of HSV-2 viral gene expression. Luciferase assays confirmed that miR-141 directly targets NLRP3, while miR-211 regulates CASP1, validating their roles as post-transcriptional repressors of inflammasome components.Discussion: These findings establish miR-141 and miR-211 as critical modulators of HSV-2-induced inflammasome activation, highlighting a novel miRNA-based regulatory mechanism. Restoring these miRNAs significantly reduces viral replication and inflammation, underscoring their potential as therapeutic targets for managing HSV-2-induced immunopathology. Future research should focus on in vivo validation and therapeutic optimization to develop miRNA-based interventions.