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Review 1: "Protective Role of RIPK1 Scaffolding against HDV-Induced Hepatocyte Cell Death and the Significance of Cytokines in Mice"

The reviewer found the study potentially informative, examining the role of RIPK1 in hepatitis delta virus (HDV) induced cell death and liver injury. While the manuscript was considered interesting, the reviewer identified several issues that need addressing.

Published onJul 02, 2024
Review 1: "Protective Role of RIPK1 Scaffolding against HDV-Induced Hepatocyte Cell Death and the Significance of Cytokines in Mice"
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Protective Role of RIPK1 Scaffolding against HDV-Induced Hepatocyte cell death and the Significance of Cytokines in Mice
Protective Role of RIPK1 Scaffolding against HDV-Induced Hepatocyte cell death and the Significance of Cytokines in Mice

Abstract Hepatitis delta virus (HDV) infection represents the most severe form of human viral hepatitis; however, the mechanisms underlying its pathology remain incompletely understood. We recently developed an HDV mouse model by injecting adeno-associated viral vectors (AAV) containing replication-competent HBV and HDV genomes. This model replicates many features of human infection, including liver injury. Notably, the extent of liver damage can be diminished with anti-TNF-α treatment. In the present study, we found that TNF-α is mainly produced by macrophages. Downstream of the TNF-α receptor (TNFR), the receptor-interacting serine/threonine-protein kinase 1 (RIPK1) serves as a cell fate regulator, playing roles in both cell survival and death pathways. In this study, we explored the function of RIPK1 and other host factors in HDV-induced cell death. We determined that the scaffolding function of RIPK1, and not its kinase activity, offers partial protection against HDV-induced apoptosis. A reduction in RIPK1 expression in hepatocytes through Cas9-mediated gene editing significantly intensifies HDV-induced damage. Contrary to our expectations, the protective effect of RIPK1 was not linked to TNF-α or macrophage activation, as their absence did not alter the extent of damage. Intriguingly, in the absence of RIPK1, macrophages confer a protective role. However, in animals unresponsive to type-I IFNs, RIPK1 downregulation did not exacerbate the damage, suggesting RIPK1’s role in shielding hepatocytes from type-I IFN-induced cell death. Interestingly, while the damage extent is similar between IFNAR KO and WT mice in terms of transaminase elevation, their cell death mechanisms differ. In conclusion, our findings reveal that HDV-induced type-I IFN production is central to inducing hepatocyte death, and RIPK1’s scaffolding function offers protective benefits. Thus, type-I IFN together with TNF-α, contribute to HDV-induced liver damage. These insights may guide the development of novel therapeutic strategies to mitigate HDV-induced liver damage and halt disease progression.Author summary Hepatitis D is the most aggressive form of viral hepatitis. Our manuscript underscores the complexity of HDV-induced liver damage, where both viral and host factors play significant roles. Previously, we demonstrated that pharmacological inhibition of TNF-α reduced HDV-induced liver damage. This result was corroborated in the present study using TNF-α-deficient mice. Moreover, we reported that the expression of the HDV antigen might have a cytotoxic effect, and HDV replication induces a strong activation of the innate immune system, accompanied by a substantial production of IFN-β. In this study, we discovered that RIPK1, a molecule described as a cell fate modulator acting downstream of TNF-α, plays a protective role during HDV replication. Contrary to our expectations, neither TNF-α nor macrophages, the primary producers of TNF-α, contributed to this protective effect. Instead, it seems type I IFN was involved. Interestingly, the role of type I IFN in HBV-induced liver damage has recently been proposed. Furthermore, our data reveals that several mechanisms of hepatocyte death are at play simultaneously during HDV replication, with apoptosis being one of them. Additional studies are needed to identify other mechanisms involved. Finally, these findings suggest that therapies targeting TNF-α and type-I IFN, or those increasing RIPK1 levels, might be effective in preventing or treating HDV-induced liver damage.

RR:C19 Evidence Scale rating by reviewer:

  • Potentially informative. The main claims made are not strongly justified by the methods and data, but may yield some insight. The results and conclusions of the study may resemble those from the hypothetical ideal study, but there is substantial room for doubt. Decision-makers should consider this evidence only with a thorough understanding of its weaknesses, alongside other evidence and theory. Decision-makers should not consider this actionable, unless the weaknesses are clearly understood and there is other theory and evidence to further support it.


Review: Manuscript by Camps and colleagues examines the potential role of RIPK1 in hepatitis delta virus induced cell death and liver injury. The authors use acute knockout of RIPK1 in combination with HDV infection and conclude that RIPK1 scaffolding role provides protective function from HDV mediated liver injury. They use RIPK1 inhibitor Nec1 to explore the role of RIPK1 kinase activity and show that this activity is not critical for HDV mediated liver injury.

The manuscript is potentially interesting but there are several issues that need to be addressed:

  • The authors should use caspase inhibitors zVAD or emricasan to investigate further the nature of cell death and confirm it is in fact apoptosis. RIPK1 can mediate apoptosis and necroptosis and thus additional tools are needed to differentiate various pathways. If caspase inhibition shows to be effective, there is also a possibility that pyroptosis could be involved and that can be addressed with knocking our caspase 1 and/or caspase-11 or by using NLRP3 inhibitor MCC950.

  • Alternatively, if RIPK1 is not required for HDV mediated cell death and liver injury, is it TRADD? Or maybe even Bcl-2 anti-apoptotic proteins.

  • The main scaffolding function of RIPK1 is activation of NF-kB and MAPKs. Thus, the authors should check if HDV infection activate these pathways in vivo and if RIPK1 ablation blocks their activation and contributes to cell death and tissue in jury in that manner.

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