The Many Faces of RIPK3: What About NASH?

Necroptosis is a recently described mode of programmed cell death which is morphologically similar to necrosis and occurs in certain cells in the context of death receptor signaling and when caspases (+/− cIAPs) are inhibited (1). The pathway involves RIPK1 dependent activation of RIPK3 which then phospho-activates MLKL. P-MLKL oligomerizes and mediates plasma membrane permeabilization. Many studies have attempted to elucidate the role of this pathway in various models of liver disease and the field is wrought with controversies and uncertainties. Some of these controversies are accounted for by strain differences in controls and knockout mice, the lack of littermate controls (genetic heterogeneity), the lack of specificity of necrostatin-1 (RIPK1 kinase inhibitor) (2), the lack of specificity of most if not all commercially available antisera (3), the uncertainty about cell type accounting for the components of the pathway (hepatocytes or non-parenchymal cells) and lastly, the necroptosis independent roles of RIPK1 and RIPK3. Furthermore, unlike apoptosis, currently there is no way to specifically identify necroptotic cell death in histologic section or serum samples. A major recent advance in the field has been the recognition that RIPK1 and RIPK3 have roles in inflammation, independent of necroptosis and mediation of cell death (1). An example is the impairment of production of cytokines (TNF and IFN-γ) by RIPK3-deficient NKT cells leading to attenuated liver injury from Concanavalin A (4).

The study of Roychowdhury and coworkers in this issue of Hepatology addresses the potential role of RIPK3 in high fat diet (HFD) model of NAFLD (5). The major finding is that steatohepatitis was markedly worsened in RIPK3−/− mice largely as a consequence of augmented hepatocyte apoptosis. Hepatocyte apoptosis is widely recognized as a major contributor to this disease with many of its features dependent on saturated fatty acid-induced lipoapoptosis, contributing to subsequent inflammation and fibrosis. Interestingly, the findings reported here in HFD model are the opposite of what was described by the same group in the alcohol model where the authors showed protection in RIPK3 −/− mice (6). This underscores that despite pathologic similarities, fundamental pathophysiologic differences may mediate liver injury in the two diseases. Curiously despite these contrary findings, in both the alcohol fed mice and the HFD fed mice an increased expression of RIPK3 was detected by immunostaining using the same polyclonal RIPK3 antibody.

Activated RIPK1 and RIPK3 can trigger apoptosis or necroptosis depending on the context. RIPK3D161N and RIPK3 small molecule inhibitors promote apoptosis which is believed to be due to induction of a conformational change in RIPK3 which engages RIPK1 to activate caspase 8 and cause apoptosis (active RIPK1 not required) (7). Thus, the use of RIPK3 inhibitors is problematic and can only be considered with concomitant apoptosis inhibitors (such as caspase inhibitors). It would be of interest to examine the effect of RIPK3 inhibitors in the HFD model to see if the same results are obtained. Global knockout of RIPK3 differs from RIPK3D161N and small molecule inhibitors in not inducing spontaneous apoptosis. In fact, RIPK3 global knockout mice, which are resistant to necroptosis, exhibit no phenotype. The mechanism for the enhanced apoptosis observed by Roychowdhury and colleagues in the RIPK3 −/− mice on a HFD is uncertain. The authors attempt to exclude a cell autonomous effect using AML12 cells but this cannot be considered definitive as these cells are not primary hepatocytes. To gain understanding of the mechanism of enhanced apoptosis, future studies will need to examine the expression of the death receptor complex components (cIAP, FLIP, xIAP, TRAF2, RIPK1, etc.) as well as the pro and anti-apoptotic BCL2 family members in RIPK3 −/− mice on a high fat diet. If the increased hepatocellular apoptosis is indeed not directly determined by the absence of RIPK3 in hepatocytes, a non-parenchymal anti-inflammatory role of RIPK3, either necroptosis dependent or independent, must be considered. Interestingly, although necroptosis is generally thought of as immunogenic, some research has suggested that necroptosis of immune cells actually lessens in vivo cytokine production so that inhibition of necroptosis can be pro-inflammatory (8).

It is of interest that the chow fed wild type mice in the present study expressed no RIPK3 or pMLKL but after HFD feeding showed increased expression of both proteins. However, despite the increased expression of these necroptosis proteins, the mice had surprisingly minimal inflammation, ALT and injury. Thus, one cannot conclude that the increased staining or expression of these proteins represents bonafide necroptosis. Others have shown that diet induced steatohepatitis is abrogated in hepatocytes from conditional caspase 8 deletion with no switch to necroptosis (9). Therefore, the significance of the increased expression of RIPK3 and pMLKL could reside in either dampening non-parenchymal cell inflammation or hepatocyte apoptosis (a pro-survival role for RIPK3 in this context). Based on the data presented, one cannot conclude that necroptosis is occurring. Furthermore, since the authors had obtained MLKL−/− mice with the purpose of having a negative control for immunostaining, we look forward to learning if these mice will phenocopy RIPK3−/− mice in this model.

Overall Roychowdhury et al present novel findings which add to the caution raised about the use of RIPK3 inhibitors and show that exclusion of RIPK3 and interference with necroptosis is not therapeutic in NASH. The phenotyping and characterization of steatohepatitis, glucose and whole body energy metabolism are superbly conducted. However, as the authors themselves point out, some caution is needed about the strain differences between their Jackson wild type controls (C57BL6/j) and the Genentech RIPK3−/− mice which are generated on a C57BL6/n background (communication with Kim Newton, Genentech). The j substrain is more resistant to steatohepatitis which may be due to a natural mutation in the mitochondrial enzyme nicotinamide nucleotide transhydrogenase (NNT) (10). Though one might predict that the effect of this mutation would be to impair antioxidant defense by limiting NADPH production, paradoxically, the j substrain has a compensatory response which makes it less susceptible to various injuries including acetaminophen and high fat diet induced steatohepatitis (10, 11). Although the authors do not comment on the substrain of RIPK3−/− mice used in this paper, the RIPK3−/− mice from their previous publication was backcrossed to a C57BL6/j substrain (6). Regardless, there are always limitations when not using littermates and the potential pitfalls of this was recently addressed in the editor’s comments of the journal (12).

One important finding in this study is that RIPK3−/− mice exhibit basal insulin resistance and enhanced sensitivity to HFD-induced worsening of insulin resistance, glucose tolerance and obesity as well as steatohepatitis. The mechanism for this basal insulin resistance is not known and it will be very important to elucidate this so as to better understand the current findings. We appreciate that the current study, though containing a very large amount of data, is just the beginning of the work that the authors will undoubtedly continue to pursue as conditional knockouts and improved reagents become available. We anticipate much progress in this important area of research.