Nippostrongylus brasiliensis and herpes simplex virus-2 co-infections: Impact on female genital tract microRNA and immune profiles, and potential role in cervical carcinogenesis.

Abstract

Background: Co-infections involving soil-transmitted helminths (STHs) and viral pathogens such as herpes simplex virus type 2 (HSV-2) remain poorly characterized, despite their overlapping prevalence in sub-Saharan Africa and other resource-poor regions. The immunomodulatory effects of STHs and the oncogenic potential of HSV-2 raise pertinent questions about whether STHs may alter host immunity and susceptibility to HSV-2 and influence molecular pathways associated with cervical cancer. MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression, which play crucial roles in immune modulation, inflammation, and cancer-related pathways. This study aimed to characterize miRNA and mRNA expression profiles in the murine female genital tract (FGT) following single and co-infection with Nippostrongylus brasiliensis (Nb), a murine hookworm, and HSV-2, to elucidate potential molecular mechanisms by which helminth-induced immunoregulation may influence HSV-2 immunity and pathways implicated in cervical cancer. Methods: A comprehensive literature review was first conducted to assess existing knowledge on STH/HSV-2 co-infections and highlighted the biological plausibility and epidemiological significance of STH/HSV-2 co-infections and informed the conceptual framework for this investigation. The review also emphasised the potential role of miRNAs in regulating immune and disease pathways in the context of STH/HSV-2 co-infections. Experimentally, female BALB/c mice were either singly infected with Nb or HSV-2, co-infected with both pathogens, or left uninfected. Total RNA was extracted from FGT tissues and high-throughput next-generation sequencing was used to characterise miRNA and mRNA expression profiles in the FGT. Differential expression analysis was conducted using edgeR and limma packages in R. Ingenuity Pathway Analysis (IPA) was used to predict canonical pathways, biological functions, and to identify key immune and cancer-related pathways associated with differentially expressed (DE) miRNAs. Selected DE miRNAs were validated by RT-qPCR. RNA-sequencing (mRNA) data were functionally analysed using g: Profiler and visualized through protein–protein interaction networks constructed in Cytoscape, with hub gene and module identification via CytoHubba and MCODE. Results: MiRNA sequencing revealed eight DE miRNAs in Nb-infected FGT tissues (e.g., mmu-miR-218- 5p, mmu-miR-449a-5p, mmu-miR-497a-3p, mmu-miR-144-3p), and nine DE miRNAs in HSV- 2-infected tissues (e.g., mmu-miR-451a, mmu-miR-376a-3p, mmu-miR-205-3p, mmu-miR-103-3p), relative to uninfected controls. Notably, only one miRNA (mmu-miR-199a-5p) was DE in co-infected tissues, suggestive of helminth-induced immunomodulation and antagonism of HSV- 2-associated immune and transcriptomic responses. Several DE miRNAs identified have established roles in immune regulation and carcinogenesis within the female reproductive tract. IPA revealed enrichment of immune-related pathways such as neutrophil degranulation, interleukin (IL)-4 and IL-13 signaling, natural killer cell signaling, and ISGylation signaling. Cancer was predicted as a significantly enriched disease, particularly in the co-infected group. Complementary mRNA profiling demonstrated 368 DE genes in Nb-infected tissues (356 upregulated, 12 downregulated), with significant expression changes in Th2-related immune genes including C-C motif chemokine ligand 11 (Ccl11), C-C chemokine receptor type 2 (Ccr2), and CX3C chemokine receptor 1 (Cx3cr1). HSV-2-infected tissues yielded 140 DE genes (121 upregulated, 19 downregulated), with alterations in genes implicated in immune function [e.g., low-density lipoprotein receptor (Ldlr), calcium/calmodulin-dependent protein kinase ID (Camk1d), LDL Receptor Related Protein 8 (Lrp8), ectopic P-granules autophagy protein 5ho molog (Epg5)] and in cell cycle and sterol biosynthesis pathways. In contrast, co-infectedti ssues did not exhibit significant differential gene expression compared to uninfected controls, further supporting the potential immunomodulatory effect of Nb on anti-HSV-2 immune and transcriptomic responses. Conclusion: This study provides the first comprehensive analysis of miRNA and mRNA expression profiles in the murine FGT following Nb and HSV-2 single and co-infections, revealing distinct transcriptional and post-transcriptional signatures associated with helminth and viral infection. The suppression of transcriptomic responses during co-infection underscores the potential of helminth-mediated immunomodulation to alter anti-HSV-2 immunity and its potential to contribute to inflammation- and cancer-related pathways. These findings fill a critical gap in understanding the molecular interplay between STHs and HSV-2 and provide the groundwork for future mechanistic studies, including functional validation and translational research. The results are relevant for diagnostics, vaccine strategies, integrated disease control in co-endemic regions, and in understanding the potential links between co-infection and cervical cancer risk.