👤 Killian E Vlaming

Persistent monocyte activation and altered cytokine responses are reported in PWH despite ART. How prior HIV-1 infection status and timing of ART initiation relate to monocyte pattern-recognition receptor crosstalk between TLR8 and RLRs remains uncertain. We conducted a comparative cohort study in adult males enrolled from two Dutch HIV-cohorts. Participants included HIV-negative participants, PWH who initiated ART during chronic HIV infection, and PWH who initiated ART during acute HIV infection, with sampling at 24 and 156 weeks after ART initiation for the acute group. PBMCs were stimulated with an RLR agonist, a TLR8 agonist, or both. Monocyte surface markers were assessed by flow cytometry and pro-inflammatory cytokines were analysed with qPCR and ELISA. Across groups, RLR stimulation induced IL-12p70 and IL-27, TLR8 stimulation induced IL-6 and IL-12p70 and combined TLR8 + RLR co-stimulation synergistically increased IL-12p70 and IL-27 while restricting IL-6. Compared with controls, CHI showed reduced IL-12p70 and IL-27 and higher IL-6. In AHI at 24 weeks, cytokine patterns and co-stimulation effects resembled HIV-negative participants; by 156 weeks, responses were attenuated and approximated CHI. In this male cohort, TLR8-RLR crosstalk was preserved early after ART initiation during acute infection but diminished over time, approaching profiles observed in chronically treated infection. These observations emphasise a potential early window after ART initiation for interventions aiming to preserve monocyte function and motivate studies to characterise underlying mechanisms. Funding for this study was obtained through a ZonMW/Aidsfonds grant NL4Cure: Bridging shock and kill strategies (446002508). Show less

The eukaryotic genome is broadly transcribed by RNA polymerase II (RNAPII) to produce protein-coding messenger RNAs (mRNAs) and a repertoire of noncoding RNAs (ncRNAs). Although RNAPII is very processive during mRNA transcription, it terminates rapidly during synthesis of many ncRNAs, particularly those that arise opportunistically from accessible chromatin at gene promoters or enhancers. The divergent fates of mRNA versus ncRNA species raise many questions about how RNAPII and associated machineries discriminate functional from spurious transcription. Restrictor, comprised of the RNA binding protein ZC3H4 and RNAPII-interacting protein WDR82, has been implicated in restraining the expression of ncRNAs. However, the determinants of Restrictor specificity and the mechanism of transcription suppression remain unclear. Here, we investigate Restrictor using unbiased sequence screens and rapid protein degradation followed by nascent RNA sequencing. We found that Restrictor promiscuously suppresses early elongation by RNAPII, but this activity is blocked at most mRNAs by the presence of a 5' splice site. Consequently, Restrictor is a critical determinant of transcription directionality at divergent promoters and prevents transcriptional interference. Mechanistically, we show that rather than terminating RNAPII directly, Restrictor acts by reducing the rate of transcription elongation, rendering RNAPII susceptible to early termination by other machineries. Show less

The eukaryotic genome is broadly transcribed by RNA polymerase II (RNAPII) to produce protein-coding messenger RNAs (mRNAs) and a repertoire of non-coding RNAs (ncRNAs). Whereas RNAPII is very processive during mRNA transcription, it terminates rapidly during synthesis of many ncRNAs, particularly those that arise opportunistically from accessible chromatin at gene promoters or enhancers. The divergent fates of mRNA versus ncRNA species raise many questions about how RNAPII and associated machineries discriminate functional from spurious transcription. The Restrictor complex, comprised of the RNA binding protein ZC3H4 and RNAPII-interacting protein WDR82, has been implicated in restraining the expression of ncRNAs. However, the determinants of Restrictor targeting and the mechanism of transcription suppression remain unclear. Here, we investigate Restrictor using unbiased sequence screens, and rapid protein degradation followed by nascent RNA sequencing. We find that Restrictor promiscuously suppresses early elongation by RNAPII, but this activity is blocked at most mRNAs by the presence of a 5' splice site. Consequently, Restrictor is a critical determinant of transcription directionality at divergent promoters and prevents transcriptional interference. Finally, our data indicate that rather than directly terminating RNAPII, Restrictor acts by reducing the rate of transcription elongation, rendering RNAPII susceptible to early termination by other machineries. Show less