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Background MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate gene expression in various biological fluids. The aim of our study was to characterize the expression profile, in CSF, of several human (hsa) and HCMV-encoded miRNAs, primarily involved in the regulation of vascular inflammation, and to compare these profiles with their expression in plasma. Correlation with CSF and plasma levels of IL-1β b, IL-6, IL-8 and TNF-α was also investigated.Methods Ten paired CSF-plasma samples from ART-treated PWH, co-infected with HCMV, were analyzed for the expression of 8 human miRNAs and 14 HCMV-encoded miRNAs, using miRCURY LNA RT Kit and UniSp6 spike-in. Quantitative PCR was then performed using specific miRCURY miRNA PCR primers. MiRNA expression levels were calculated using ΔCt, with UniSp6 Ct as reference. CSF and plasma levels of IL-1β, IL-6, IL-8 and TNF- α were quantified by ELISA, with 5 plasma samples from healthy donors (HD) as controls.Results The subjects were 9 males and 1 female, with a median (IQR) age of 51.5 (46.0-67.8). Median (IQR) HIV-1 RNA were 2.87 (1.39-3.69) and 1.95 (1.46-4.59) Log copies/ml in CSF and plasma, respectively. HCMV DNA was detected in 1 CSF and in 4 plasma samples. All human miRNAs were more highly expressed in plasma than CSF, and 5 out of 8 were highly expressed in the plasma of PWH compared to HD ( table 1A). Hsa-miR20b-5p was detected in only 2 CSF samples, while hsa-150-3p and hsa-155-5p only in 3 CSF. Among HCMV-encoded miRNAs, hcmv-US33-5p and hcmv-UL-70-3p were consistently expressed. Hcmv-UL22A-5p was detected in all plasma and only in 1 CSF samples. Other HCMV-encoded miRNAs were variably expressed, with hcmv-UL148D more frequently detected. In a subject with HCMV-related encephalitis, all HCMV-encoded miRNAs, except hcmv-miR-US4-5p, were found in CSF. Plasma cytokine levels were higher in PWH compared to HD. IL-8 and TNF-α were predominantly expressed at higher levels in CSF and plasma, respectively. IL-1β was also significantly higher in plasma than in CSF (table 1B). IL-8 showed a statistically significant positive correlation with all human miRNAs, while TNF-α showed a statistically significant negative correlation. IL-1β levels were negatively correlated with 7 out of 8 human miRNAs. Conversely, HCMV-encoded hcmv-US33-5p and hcmv-UL-70-3p showed a significant positive correlation with IL-1β and TNF-α (table 1C). No correlations were observed between overall miRNAs, cytokines and HIV-1 RNA levels, calculated by combining the two compartments.Conclusion Human and HCMV-encoded miRNAs display distinct expression patterns in plasma and CSF of ART-treated PWH, and show differential associations with inflammatory cytokines. These preliminary findings suggest a potential interplay between host and viral miRNAs in the inflammatory milieu of treated HIV infection. Larger studies are needed to confirm these observations and clarify their clinical significance.Abstract OC17 Table 1Comparison of human miRNA expression in CSF and plasma of PWH and plasma of HD (A); cytokine levels in CSF and plasma of PWH and plasma HD (B); correlation between human and HCMV-encoded miRNA expression and plasma cytokines (C). Hsa-miR is used to describe human miRNAs, while hcmv refers to human cytomegalovirusmiRNAs