Immunomodulation of NK cells by Ribavirin is driven by pSTAT-4 activation with increased IFN-γ secretion in HEV

Background Hepatitis E virus (HEV) is one of the main causes for acute hepatitis globally. In most cases the infection is asymptomatic and self-limiting. Nevertheless, in some patient groups, such as immunocompromised organ transplant recipients, it can lead to severe courses. Natural Killer (NK) cells are an important part of the innate immune response and represent a first line of defense against viral pathogens by producing the antiviral cytokine IFN-γ and by natural cytotoxicity. Here we asked which impact NK cells have on a HEV infection in the context of Ribavirin (RBV), the current treatment for a chronic HEV infection.

Methods The human hepatoma cell line HepaRG was inoculated with a full-length HEV (MOI 0.5). After 7 days of culture the cells were co-cultivated with PBMCs from healthy donors for 24h at an E:T ratio of 1:1 or with RBV at a concentration of 500µM. The viral replication was measured by qPCR and NK cells were analyzed by flow cytometry.

Results Both the co-culture of PBMCs and the treatment with sole RBV decreased viral loads, however the combination had a synergistic effect. NK cells stimulated with RBV showed an increased expression of the activation marker CD38 (3439/3837; p<0.0001) as well as the activatory receptor NKp46 (3309/3501; p=0.0139) and a decrease of the inhibitory receptor TIGIT (1414/743; p=0.0005). Looking at NK cell functions, we found a reduction in cytotoxicity as assessed by expression of TRAIL (472/415; p=0.0087) and CD107a degranulation (258/212; p=0.0002), while the production of IFN-γ was significantly increased (1020/1394; P<0.0001). To identify the underlying mechanism, we investigated different cytokine stimulations such as IL-12, IL-15 and IL-18. Compared to the unstimulated controls the combination of IL-12/IL-15 (change 415; p<0.0001) and sole IL-12 (change 46; P=0.0127) were affected by RBV treatment while stimulation with IL-15, IL-18 or combined IL-12/IL-18 were not. Indicating, that RBV is acting on a distinct signaling pathway. Since expression of the IL-12Rβ1- and -Rβ2-subunits were not upregulated, we analyzed downstream events by phosflow staining. We found an increase in pSTAT-4 expression (450/478; p=0.0004) while other transcription factors such as pSTAT-1 and pSTAT-3 were not affected.

Conclusion In the context of an in-vitro HEV infection, RBV has an immunomodulatory effect on NK cells by increasing pSTAT-4 expression and subsequently enhanced IFN-γ production.

Publication History

Article published online:
04 January 2021