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The activation phenotypes and functional changes in monocyte subsets during hepatitis C virus (HCV) elimination in HIV/HCV-coinfected patients were evaluated. Twenty-two HIV/HCV-coinfected patients on suppressive combination antiretroviral treatment (cART) achieving HCV elimination after direct-acting antiviral (DAA) therapy and 10 HIV-monoinfected patients were included. The activation phenotype (10 markers) and polyfunctionality (intracellular interleukin-1α [IL-1α], IL-1β, IL-6, IL-8, tumor necrosis factor alpha [TNF-α], and IL-10 production) in three monocyte subsets (classical, intermediate, and nonclassical) were evaluated by flow cytometry before and at the end of treatment. Cell-associated HIV DNA levels were assayed by droplet digital PCR. After HCV clearance, there was a significant increase in classical monocyte and decreases in intermediate and nonclassical monocyte levels. The levels of the activation markers CD49d, CD40, and CX3CR1 were decreased after treatment in the monocyte subsets, reaching the levels in HIV-monoinfected patients. After lipopolysaccharide (LPS) stimulation, although polyfunctionality significantly decreased in intermediate and nonclassical monocytes, some combinations, such as the IL-1α− (IL-1α-negative) IL-1β− IL-6+ (IL-6-producing) IL-8− TNF-α− IL-10− combination, were remarkably increased at the end of treatment compared to the control group. Cell-associated HIV DNA levels correlated with activation markers before but not after treatment. HCV clearance after DAA treatment in patients on cART exerts an anti-inflammatory profile on monocyte subsets, activation phenotypes, and polyfunctionality. However, there is not a complete normalization compared with HIV-monoinfected patients., This work was supported by the Instituto de Salud Carlos III (research contracts CPII014/00025 to E.R.-M., CP19/00159 to A.G.-V., FI17/00186 to M.R.J.-L., and FI14/00431 to L.T.-D. and research projects PI16/00684 and PI19/01127 to E.R.-M.) and the Red Temática de Investigación Cooperativa en SIDA (RD16/0025/0020 and RD16/0025/0019), which is included in the Acción Estratégica en Salud, Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica, 2008 to 2011 and 2013 to 2016, Instituto de Salud Carlos III, Fondos FEDER. E.R.-M. was supported by the Consejería de Salud y Bienestar Social of the Junta de Andalucía through the Nicolás Monardes program (C-0032/17).

Plasmacytoid dendritic cells (pDCs) are innate immune cells with high antiviral activity triggered by Toll-like receptor 7 (TLR-7) and TLR-9 stimulation. Moreover, they are important mediators between innate and adaptive immunity. Although nowadays there is available an effective therapeutic arsenal against hepatitis C virus (HCV), a protective vaccine is not available. We have analyzed the pDCs’ response to HCV infection in a hepatitis C virus (HCV)-Huh7.5 virus-cell system, which allows completion of the virus infectious cycle. pDCs were cocultured following human immunodeficiency virus (HIV) aldrithiol-2 (AT-2 [TLR-7 agonist]) inactivation and CpG (TLR-9 agonist) stimulation. We employed three virus derivatives—wild-type Jc1, interferon (IFN)-resistant virus IR, and high-replicative-fitness virus P100—in order to explore additional IFN-α-related virus inhibition mechanisms. pDCs inhibited HCV infectivity and replication and produced IFN-α. After TLR-7 and TLR-9 stimulation, inhibition of infectivity and IFN-α production by pDCs were enhanced. TLR-7 stimulation drove higher TNF-related apoptosis-inducing ligand (TRAIL) expression in pDCs. Additionally, TLR-7- and TLR-9-stimulated pDCs exhibited a mature phenotype, improving the antigen presentation and lymph node homing-related markers. In conclusion, pDCs could serve as a drug target against HCV in order to improve antiviral activity and as an enhancer of viral immunization. IMPORTANCE We implemented a coculture system of pDCs with HCV-infected hepatoma cell line, Huh7.5. We used three HCV derivatives in order to gain insight into pDCs’ behavior against HCV and associated antiviral mechanisms. The results with this cell coculture system support the capacity of pDCs to inhibit HCV replication and infectivity mainly via IFN-α, but also through additional mechanisms associated with pDC maturation. We provided evidence that TLR agonists can enhance antiviral pDCs’ function and can induce phenotypic changes that may facilitate the interplay with other immune cells. These findings suggest the possibility of including TLR agonists in the strategies of HCV vaccine development.