Although necrosis in the acetaminophen (APAP) model is known to be regulated by c-Jun NH2- terminal kinase, (JNK) through interaction with mitochondria, the role of necroptosis through receptor interacting proteins 1 and 3 (RIPK1 and RIPK3) has also been suggested. Our aim was to determine the relationship between these two mechanisms of cell death. To verify the participation of RIPK1, we used antisense knockdown and confirmed protection comparable to the RIPK1 inhibitor, necrostatin in vivo and in vitro. However, we found no evidence that RIPK3 is expressed in primary mouse hepatocytes under basal conditions or after APAP and RIPK3−/− mice were not protected. RIPK3 was exclusively expressed in nonparenchymal cells. RIPK1 knockdown protected RIPK3−/− mice to the same extent as wild type mice, underscoring the independent role of RIPK1. We confirmed necroptosis is not involved in APAP toxicity by using mixed lineage kinase domain-like protein (MLKL) −/− mice, which were not protected from APAP. Next we addressed if there is interplay between RIPK1 and JNK. RIPK1 knockdown decreased the level of JNK activation and translocation to mitochondria and abrogated subsequent translocation of dynamin-related protein-1 (Drp1). Interestingly, APAP induced the translocation of RIPK1 to mitochondria which was unaffected by the knockdown of the mitochondrial JNK docking protein, Sh3 homology3 binding protein5 (Sab). Conclusion RIPK1 participates in APAP induced necrosis upstream of JNK activation while RIPK3 and MLKL are dispensable, indicating that necroptosis does not contribute to APAP-induced necrosis and RIPK1 has a unique, independent role.

Recent studies have demonstrated immunologic dysfunction in severely ill COVID-19 patients. We use single-cell RNA sequencing (scRNA-seq) to analyze the transcriptome of peripheral blood mononuclear cells (PBMC) from healthy (n=3) and COVID-19 patients with moderate disease (n = 5), acute respiratory distress syndrome (ARDS, n = 6), or recovering from ARDS (n = 6). Our data reveal transcriptomic profiles indicative of defective antigen presentation and interferon responsiveness in monocytes from ARDS patients, which contrasts with higher responsiveness to interferon signaling in lymphocytes. Furthermore, genes involved in cytotoxic activity are suppressed in both NK and CD8 lymphocytes, and B cell activation is deficient, which is consistent with delayed viral clearance in severely ill COVID-19 patients. Our study demonstrates that COVID-19 patients with ARDS have a state of immune imbalance in which dysregulation of both the innate and adaptive immune responses may be contributing to a more severe disease course.

Coronavirus disease 2019 (COVID-19) has quickly become the most serious pandemic since the 1918 flu pandemic. In extreme situations, patients develop a dysregulated inflammatory lung injury called acute respiratory distress syndrome (ARDS) that causes progressive respiratory failure requiring mechanical ventilatory support. Recent studies have demonstrated immunologic dysfunction in severely ill COVID-19 patients. To further delineate the dysregulated immune response driving more severe clinical course from SARS-CoV-2 infection, we used single-cell RNA sequencing (scRNAseq) to analyze the transcriptome of peripheral blood mononuclear cells (PBMC) from hospitalized COVID-19 patients having mild disease (n = 5), developing ARDS (n = 6), and recovering from ARDS (n = 6). Our data demonstrated an overwhelming inflammatory response with select immunodeficiencies within various immune populations in ARDS patients. Specifically, their monocytes had defects in antigen presentation and deficiencies in interferon responsiveness that contrasted the higher interferon signals in lymphocytes. Furthermore, cytotoxic activity was suppressed in both NK and CD8 lymphocytes whereas B cell activation was deficient, which is consistent with the delayed viral clearance in severely ill COVID-19 patients. Finally, we identified altered signaling pathways in the severe group that suggests immunosenescence and immunometabolic changes could be contributing to the dysfunctional immune response. Our study demonstrates that COVID-19 patients with ARDS have an immunologically distinct response when compared to those with a more innocuous disease course and show a state of immune imbalance in which deficiencies in both the innate and adaptive immune response may be contributing to a more severe disease course in COVID-19.

Receptor-interacting protein 1(RIPK1) has an essential role in the signaling pathways triggered by death receptors through activation of NF-κB and regulation of caspase-dependent apoptosis and RIPK3/mixed lineage kinase domain-like protein(MLKL)-mediated necroptosis. We examined the effect of RIPK1 antisense knockdown on the immune-mediated liver injury in C57BL/6 mice caused by α-galactosylceramide (αGalCer), a specific activator for invariant NKT cells. We found that knockdown of RIPK1 markedly exacerbated αGalCer-mediated liver injury and induced lethality. This was associated with increased hepatic inflammation and massive apoptotic death of hepatocytes as indicated by TUNEL staining and caspase-3 activation. Pretreatment with either zVAD.fmk, a pan-caspase inhibitor, or neutralizing Abs against TNF, almost completely protected against the exacerbated liver injury and lethality. Primary hepatocytes isolated from RIPK1 knockdown mice were sensitized to TNF-induced cell death that was completely inhibited by adding zVAD.fmk. Unexpectedly, the exacerbated liver injury was not due to impaired hepatic NF-κB activation in terms of IκBα phosphorylation and degradation in both in vivo and in vitro studies. Lack of RIPK1 kinase activity by either pretreatment with necrostatin-1, a RIPK1 kinase inhibitor, or in the RIPK1 kinase-dead knock-in (RIPK1D138N) mice, did not exacerbate αGalCer-mediated liver injury. Furthermore, both RIPK3 and MLKL knockout mice behaved similarly as wild type control mice in response to αGalCer with or without knockdown of RIPK1, excluding a switch to RIPK3/MLKL-mediated necroptosis. Our findings for the first time revealed a critical kinase-independent platform role of RIPK1 in protecting against TNF/caspase-dependent apoptosis of hepatocytes in immune-mediated liver injury.

Radixin, the dominant ezrin-radixin-moesin (ERM) protein in hepatocytes, has two important binding domains: an NH(2)-terminal region that binds to plasma membrane and a COOH-terminal region that binds to F-actin after a conformational activation by phosphorylation at Thr564. The present studies were undertaken to investigate the cellular changes in expression of radixin in WIF-B cells and to assess radixin distribution and its influence on cell polarity. We used a recombinant adenoviral expression system encoding radixin wild-type and Thr564 mutants fused to cyan fluorescent protein (CFP), as well as conventional immunostaining procedures. Functional analyses were characterized quantitatively. Similar to endogenous radixin, adenovirus-infected radixin-CFP-wild type and nonphosphorylatable radixin-CFP-T564A were found to be expressed heavily in the compartment of canalicular membrane vacuoles, typically colocalizing with multidrug resistance-associated protein 2 (Mrp-2). Expression of radixin-CFP-T564D, which mimics constant phosphorylation, was quite different, being rarely associated with canalicular membranes. The WIF-B cells were devoid of a secretory response, T567D radixin became predominantly redistributed to the basolateral membrane, usually in the form of dense, long spikes and fingerlike projections, and the altered cell polarity involved changes in apical membrane markers. Differences in polar distribution of radixin suggest a role for the linker protein in promoting formation and plasticity of membrane surface projections and also suggest that radixin might be an organizer and regulator of Mrp-2 and cell polarity in hepatocytes.

The role of lysosomes in acetaminophen (APAP) hepatotoxicity is poorly understood. Here, we investigated the impact of genetic and drug-induced lysosomal cholesterol (LC) accumulation in APAP hepatotoxicity. Acid sphingomyelinase (ASMase)−/− mice exhibit LC accumulation and higher mortality after APAP overdose compared to ASMase+/+ littermates. ASMase−/− hepatocytes display lower threshold for APAP-induced cell death and defective fusion of mitochondria-containing autophagosomes with lysosomes, which decreased mitochondrial quality control. LC accumulation in ASMase+/+ hepatocytes caused by U18666A reproduces the susceptibility of ASMase−/− hepatocytes to APAP and the impairment in the formation of mitochondria-containing autolysosomes. LC extraction by 25-hydroxycholesterol increased APAP-mediated mitophagy and protected ASMase−/− mice and hepatocytes against APAP hepatotoxicity, effects that were reversed by chloroquine to disrupt autophagy. The regulation of LC by U18666A or 25-hydroxycholesterol did not affect total cellular sphingomyelin content or its lysosomal distribution. Of relevance, amitriptyline-induced ASMase inhibition in human hepatocytes caused LC accumulation, impaired mitophagy and increased susceptibility to APAP. Similar results were observed upon glucocerebrosidase inhibition by conduritol β-epoxide, a cellular model of Gaucher disease. These findings indicate that LC accumulation determines susceptibility to APAP hepatotoxicity by modulating mitophagy, and imply that genetic or drug-mediated ASMase disruption sensitizes to APAP-induced liver injury.

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Background and Aims Moesin, an ezrin/radixin/moesin family member, is involved in the regulation of cell adhesion, polarity, and migration by cross‐linking between the actin cytoskeleton and plasma membrane. The primary effector cell in hepatic fibrosis is the hepatic stellate cell (HSC), which undergoes activation during liver injury leading to increased extracellular matrix production. Approach and Results Here, we have hypothesized that moesin plays a critical role in linking the HSC cytoskeleton to the fibrogenic cascade during HSC activation. Moesin phosphorylation was up‐regulated during HSC activation and fibrogenesis. Using moesin wild‐type (WT) and mutant constructs (phosphomimicking T558D and nonphosphorylatable T558A), we found that cellular motility and contraction were increased in moesin WT‐infected and T558D‐infected cells, paralleled by an increase in smooth muscle α‐actin and collagen 1 expression. In contrast, overexpression of nonphosphorylatable moesin and moesin knockout (KO) decreased cellular motility and contraction. Most importantly, moesin KO led to abrogation of liver fibrosis. The mechanism of moesin's effect was a reduction in myocardin‐related transcription factor‐A and serum‐response factor (SRF)–mediated changes in the actin cytoskeleton, which in turn modulated the expression of matrix genes. Conclusions Taken together, our findings suggest that the linkage between cytoskeletal dynamics and the correlated MRTF/SRF signaling pathway has a pivotal role in HSC activation and fibrogenesis.