Trim21 deficiency in mice increases HCC carcinogenesis in a NASH context and is associated with immune checkpoint upregulation.

The global pandemic of metabolic diseases has increased the incidence of hepatocellular carcinoma (HCC) in the context of non-alcoholic steatohepatitis (NASH). The downregulation of the E3 ubiquitin ligase TRIM21 has been linked to poor prognosis in different cancers including HCC. In order to investigate the role of TRIM21 in liver cancer progression on NASH, Trim21+/+ and Trim21-/- male mice were injected with streptozotocin at the neonatal stage. The hypoinsulinemic mice were then fed with a high-fat high-cholesterol diet (HFHCD) for 4, 8 or 12 weeks. All mice developed NASH which systematically resulted in HCC progression. Interestingly, compared to the Trim21+/+ control mice, liver damage was worsened in Trim21-/- mice, with more HCC nodules found after 12 weeks on HFHCD. Immune population analysis in the spleen and liver revealed a higher proportion of CD4+PD-1+ and CD8+PD-1+ T cells in Trim21-/- mice. The liver and HCC tumors of Trim21-/- mice also exhibited an increase in the number of PD-L1+ and CD68+ PD-L1+ cells. Thus, TRIM21 limits the emergence of HCC nodules in mice with NASH by potentially restricting the expression of PD-1 in lymphocytes and PD-L1 in tumors.

RIPK1 in Liver Parenchymal Cells Limits Murine Hepatitis during Acute CCl4-Induced Liver Injury.

Some life-threatening acute hepatitis originates from drug-induced liver injury (DILI). Carbon tetrachloride (CCl4)-induced acute liver injury in mice is the widely used model of choice to study acute DILI, which pathogenesis involves a complex interplay of oxidative stress, necrosis, and apoptosis. Since the receptor interacting protein kinase-1 (RIPK1) is able to direct cell fate towards survival or death, it may potentially affect the pathological process of xenobiotic-induced liver damage. Two different mouse lines, either deficient for Ripk1 specifically in liver parenchymal cells (Ripk1LPC-KO) or for the kinase activity of RIPK1 (Ripk1K45A, kinase dead), plus their respective wild-type littermates (Ripk1fl/fl, Ripk1wt/wt), were exposed to single toxic doses of CCl4. This exposure led in similar injury in Ripk1K45A mice and their littermate controls. However, Ripk1LPC-KO mice developed more severe symptoms with massive hepatocyte apoptosis as compared to their littermate controls. A pretreatment with a TNF-α receptor decoy exacerbated liver apoptosis in both Ripk1fl/fl and Ripk1LPC-KO mice. Besides, a FasL antagonist promoted hepatocyte apoptosis in Ripk1fl/fl mice but reduced it in Ripk1LPC-KO mice. Thus, the scaffolding properties of RIPK1 protect hepatocytes from apoptosis during CCl4 intoxication. TNF-α and FasL emerged as factors promoting hepatocyte survival. These protective effects appeared to be independent of RIPK1, at least in part, for TNF-α, but dependent on RIPK1 for FasL. These new data complete the deciphering of the molecular mechanisms involved in DILI in the context of research on their prevention or cure.

Receptor-interacting protein kinase-1 ablation in liver parenchymal cells promotes liver fibrosis in murine NASH without affecting other symptoms.

Non-alcoholic steatohepatitis (NASH), a chronic liver disease that emerged in industrialized countries, can further progress into liver fibrosis, cirrhosis, and hepatocellular carcinoma. In the next decade, NASH is predicted to become the leading cause of liver transplantation, the only current interventional therapeutic option. Hepatocyte death, triggered by different death ligands, plays key role in its progression. Previously, we showed that the receptor-interacting protein kinase-1 (RIPK1) in hepatocytes exhibits a protective role in ligand-induced death. Now, to decipher the role of RIPK1 in NASH, Ripk1LPC-KO mice, deficient for RIPK1 only in liver parenchymal cells, and their wild-type littermates (Ripk1fl/fl) were fed for 3, 5, or 12 weeks with high-fat high-cholesterol diet (HFHCD). The main clinical signs of NASH were analyzed to compare the pathophysiological state established in mice. Most of the symptoms evolved similarly whatever the genotype, whether it was the increase in liver to body weight ratio, the steatosis grade or the worsening of liver damage revealed by serum transaminase levels. In parallel, inflammation markers followed the same kinetics with significant equivalent inductions of cytokines (hepatic mRNA levels and blood cytokine concentrations) and a main peak of hepatic infiltration of immune cells at 3 weeks of HFHCD. Despite this identical inflammatory response, more hepatic fibrosis was significantly evidenced at week 12 in Ripk1LPC-KO mice. This coincided with over-induced rates of transcripts of genes implied in fibrosis development (Tgfb1, Tgfbi, Timp1, and Timp2) in Ripk1LPC-KO animals. In conclusion, our results show that RIPK1 in hepatocyte limits the progression of liver fibrosis during NASH.

Switching to Regular Diet Partially Resolves Liver Fibrosis Induced by High-Fat, High-Cholesterol Diet in Mice.

The globally prevalent disease, non-alcoholic steatohepatitis (NASH), is characterized by a steatotic and inflammatory liver. In NASH patients, tissue repair mechanisms, activated by the presence of chronic liver damage, lead to the progressive onset of hepatic fibrosis. This scar symptom is a key prognostic risk factor for liver-related morbidity and mortality. Conflicting reports discuss the efficiency of dietary interventions on the reversibility of advanced fibrosis established during NASH. In the present study, the effect of dietary interventions was investigated in the outcome of the fibrosis settled in livers of C57BL/6J mice on a high-fat, high-cholesterol diet (HFHCD) for 5 or 12 consecutive weeks. Various clinico-pathological investigations, including a histological analysis of the liver, measurement of plasma transaminases, steatosis and fibrosis, were performed. To assess the effectiveness of the dietary intervention on established symptoms, diseased mice were returned to a standard diet (SD) for 4 or 12 weeks. This food management resulted in a drastic reduction in steatosis, liver injuries, inflammatory markers, hepatomegaly and oxidative stress and a gradual improvement in the fibrotic state of the liver tissue. In conclusion, our results demonstrated that dietary intervention can partially reverse liver fibrosis induced by HFHCD feeding.

Hepatocellular Carcinoma Emergence in Diabetic Mice with Non-Alcoholic Steatohepatitis Depends on Diet and Is Delayed in Liver Exhibiting an Active Immune Response.

The increase of the sedentary lifestyle and high-calorie diet have modified the etiological landscape of hepatocellular carcinoma (HCC), with a recrudescence of non-alcoholic fatty liver disease (NAFLD), especially in Western countries. The purpose of our study was to evaluate the impact of high-fat diet feeding on non-alcoholic steatohepatitis (NASH) establishment and HCC development. Streptozotocin-induced diabetic male mice were fed with high-fat-high-cholesterol diet (HFHCD) or high-fat-high-sugar diet (HFHSD) from 1 to 16 weeks. Even if liver tumors appear regardless of the high-fat diet, two distinct physiopathological patterns were evidenced, with much more severe NASH hallmarks (liver injury, inflammation and fibrosis) in diabetic mice fed with HFHCD. The mild hepatic injury, weak inflammation and fibrosis observed in HFHSD were interestingly associated with earlier emergence of more numerous liver tumors. When activated helper and cytotoxic T cells, detected by flow cytometry, infiltrated the liver of HFHCD-fed diabetic mice, a delay in the appearance of tumor nodules and a limitation of their numbers were observed, suggesting that the immune activities partly controlled tumor emergence. These data highlighted two different mouse models of HCC progression in diabetic mice depending on diet, which could be useful to evaluate new therapeutic approaches for HCC by targeting the immune response.

Combined anti-fibrotic and anti-inflammatory properties of JAK-inhibitors on macrophages in vitro and in vivo: Perspectives for scleroderma-associated interstitial lung disease.

Janus kinase (JAK) inhibitors (also termed Jakinibs) constitute a family of small drugs that target various isoforms of JAKs (JAK1, JAK2, JAK3 and/or tyrosine kinase 2 (Tyk2)). They exert anti-inflammatory properties linked, in part, to the modulation of the activation state of pro-inflammatory M1 macrophages. The exact impact of JAK inhibitors on a wider spectrum of activation states of macrophages is however still to be determined, especially in the context of disorders involving concomitant activation of pro-inflammatory M1 macrophages and profibrotic M2 macrophages. This is especially the case in autoimmune pulmonary fibrosis like scleroderma-associated interstitial lung disease (ILD), in which M1 and M2 macrophages play a key pathogenic role. In this study, we directly compared the anti-inflammatory and anti-fibrotic effects of three JAK inhibitors (ruxolitinib (JAK2/1 inhibitor); tofacitinib (JAK3/2 inhibitor) and itacitinib (JAK1 inhibitor)) on five different activation states of primary human monocyte-derived macrophages (MDM). These three JAK inhibitors exert anti-inflammatory properties towards macrophages, as demonstrated by the down-expression of key polarization markers (CD86, MHCII, TLR4) and the limited secretion of key pro-inflammatory cytokines (CXCL10, IL-6 and TNFα) in M1 macrophages activated by IFNγ and LPS or by IFNγ alone. We also highlighted that these JAK inhibitors can limit M2a activation of macrophages induced by IL-4 and IL-13, as notably demonstrated by the down-regulation of the M2a associated surface marker CD206 and of the secretion of CCL18. Moreover, these JAK inhibitors reduced the expression of markers such as CXCL13, MARCO and SOCS3 in alternatively activated macrophages induced by IL-10 and dexamethasone (M2c + dex) or IL-10 alone (M2c MDM). For all polarization states, Jakinibs with inhibitory properties over JAK2 had the highest effects, at both 1 µM or 0.1 µM. Based on these in vitro results, we also explored the effects of JAK2/1 inhibition by ruxolitinib in vivo, on mouse macrophages in a model of HOCl-induced ILD, that mimics scleroderma-associated ILD. In this model, we showed that ruxolitinib significantly prevented the upregulation of pro-inflammatory M1 markers (TNFα, CXCL10, NOS2) and pro-fibrotic M2 markers (Arg1 and Chi3L3). These results were associated with an improvement of skin and pulmonary involvement. Overall, our results suggest that the combined anti-inflammatory and anti-fibrotic properties of JAK2/1 inhibitors could be relevant to target lung macrophages in autoimmune and inflammatory pulmonary disorders that have no efficient disease modifying drugs to date.

Depletion of RIPK1 in hepatocytes exacerbates liver damage in fulminant viral hepatitis.

The protein kinase RIPK1 plays a crucial role at the crossroad of stress-induced signaling pathways that affects cell's decision to live or die. The present study aimed to define the role of RIPK1 in hepatocytes during fulminant viral hepatitis, a worldwide syndrome mainly observed in hepatitis B virus (HBV) infected patients. Mice deficient for RIPK1, specifically in liver parenchymal cells (Ripk1LPC-KO) and their wild-type littermates (Ripk1fl/fl), were challenged by either the murine hepatitis virus type 3 (MHV3) or poly I:C, a synthetic analog of double-stranded RNA mimicking viral pathogen-associated molecular pattern. Ripk1LPC-KO mice developed more severe symptoms at early stage of the MHV3-induced fulminant hepatitis. Similarly, administration of poly I:C only triggered increase of systemic transaminases in Ripk1LPC-KO mice, reflecting liver damage through induced apoptosis as illustrated by cleaved-caspase 3 labeling of liver tissue sections. Neutralization of TNF-α or prior depletion of macrophages were able to prevent the appearance of apoptosis of hepatocytes in poly I:C-challenged Ripk1LPC-KO mice. Moreover, poly I:C never induced direct hepatocyte death in primary culture whatever the murine genotype, while it always stimulated an anti-viral response. Our investigations demonstrated that RIPK1 protects hepatocytes from TNF-α secreted from macrophages during viral induced fulminant hepatitis. These data emphasize the potential worsening risks of an HBV infection in people with polymorphism or homozygous amorphic mutations already described for the RIPK1 gene.

Chlordecone potentiates auto-immune hepatitis and promotes brain entry of MHV3 during viral hepatitis in mouse models.

Chlordecone is an organochlorine used in the 1970's as a pesticide in banana plantations. It has a long half-life in the soil and can potentially contaminate humans and animals through food. Chlordecone targets, and mainly accumulates in, the liver, leading to hepatomegaly and neurological signs in mammals. Chlordecone does not cause liver injuries or any inflammation by itself at low doses, but it can potentiate the hepatotoxic effects of other chemicals and drugs. We studied the impact of chlordecone on the progression of acute hepatitis in mouse models of co-exposure to chlordecone with Concanavalin A or murine hepatitis virus type 3. We examined the progression of these two types of hepatitis by measuring hepatic transaminase levels in the serum and inflammatory cells in the liver, liver histological studies. Amplified tremors presented in the MHV3- chlordecone mouse model had led us to study the expression of specific genes in the brain. We show that chlordecone amplifies the auto-immune hepatitis induced by Concanavalin A by increasing the number of liver NKT cells, which are involved in liver damage. Chlordecone also accelerated the death of mice infected by murine hepatitis virus and enhanced the entry of the virus into the cervical spinal cord in infected mice, leading to considerable neurological damage. In conclusion, chlordecone potentiates both the Concanavalin A-induced hepatitis and brain damage caused by an hepatotropic/neurotropic virus.

RIPK1 protects hepatocytes from death in Fas-induced hepatitis.

Hepatocyte death is a central event during liver disease progression, in which immune cells play key roles by activating members of the Tumor Necrosis Factor Receptor Superfamily (TNFRSF), including TNFR1 (TNFRSF1A), Fas (TNFRSF6) and TRAIL-R2 (TNFRSF10B). Receptor Interacting Protein Kinase 1 (RIPK1) emerged as a signaling node downstream of these receptors. In the case of TNFR1, RIPK1 has been demonstrated to paradoxically serve as a scaffold to promote the survival of hepatocytes and as a kinase to kill them. To evaluate whether RIPK1 also protects hepatocytes from death in response to FasL or TRAIL, we took advantage of liver parenchymal cell-specific Ripk1 knockout mice (Ripk1 LPC-KO). We found that Ripk1 LPC-KO mice, as well as primary hepatocytes derived from them, were more susceptible to Fas-mediated apoptosis than their respective WT counterparts. Fas-induced hepatocyte death was independent of TNF-α signaling. Interestingly, while TRAIL administration did not induce hepatitis in Ripk1 LPC-KO mice or in their WT counterparts, its combination with IFN-γ only induced TNF-α dependent apoptosis in the Ripk1 LPC-KO mice. Together, our data demonstrate the protective role of RIPK1 downstream of Fas and highlight the general protective function of RIPK1 in hepatocytes exposed to inflammatory conditions, where TNF-α, FasL and/or TRAIL are present.

PARP2 deficiency affects invariant-NKT-cell maturation and protects mice from concanavalin A-induced liver injury.

Excessive or persistent inflammation and hepatocyte death are the key triggers of liver diseases. The poly(ADP-ribose) polymerase (PARP) proteins induce cell death and inflammation. Chemical inhibition of PARP activity protects against liver injury during concanavalin A (ConA)-induced hepatitis. In this mice model, ConA activates immune cells, which promote inflammation and induce hepatocyte death, mediated by the activated invariant natural killer T (iNKT) lymphocyte population. We analyzed immune cell populations in the liver and several lymphoid organs, such as the spleen, thymus, and bone marrow in Parp2-deficient mice to better define the role of PARP proteins in liver immunity and inflammation at steady state and during ConA-induced hepatitis. We show that 1) the genetic inactivation of Parp2, but not Parp1, protected mice from ConA hepatitis without deregulating cytokine expression and leucocyte recruitment; 2) cellularity was lower in the thymus, but not in spleen, liver, or bone marrow of Parp2-/- mice; 3) spleen and liver iNKT lymphocytes, as well as thymic T and NKT lymphocytes were reduced in Parp2 knockout mice. In conclusion, our results suggest that the defect of T-lymphocyte maturation in Parp2 knockout mice leads to a systemic reduction of iNKT cells, reducing hepatocyte death during ConA-mediated liver damage, thus protecting the mice from hepatitis.NEW & NOTEWORTHY The genetic inactivation of Parp2, but not Parp1, protects mice from concanavalin A hepatitis. Immune cell populations are lower in the thymus, but not in the spleen, liver, or bone marrow of Parp2-deficient mice compared with wild-type mice. Spleen and liver invariant natural killer T (NKT) lymphocytes, as well as thymic T and NKT lymphocytes, are reduced in Parp2-deficient mice.

Sibiriline, a new small chemical inhibitor of receptor-interacting protein kinase 1, prevents immune-dependent hepatitis.

Necroptosis is a regulated form of cell death involved in several disease models including in particular liver diseases. Receptor-interacting protein kinases, RIPK1 and RIPK3, are the main serine/threonine kinases driving this cell death pathway. We screened a noncommercial, kinase-focused chemical library which allowed us to identify Sibiriline as a new inhibitor of necroptosis induced by tumor necrosis factor (TNF) in Fas-associated protein with death domain (FADD)-deficient Jurkat cells. Moreover, Sib inhibits necroptotic cell death induced by various death ligands in human or mouse cells while not protecting from caspase-dependent apoptosis. By using competition binding assay and recombinant kinase assays, we demonstrated that Sib is a rather specific competitive RIPK1 inhibitor. Molecular docking analysis shows that Sib is trapped closed to human RIPK1 adenosine triphosphate-binding site in a relatively hydrophobic pocket locking RIPK1 in an inactive conformation. In agreement with its RIPK1 inhibitory property, Sib inhibits both TNF-induced RIPK1-dependent necroptosis and RIPK1-dependent apoptosis. Finally, Sib protects mice from concanavalin A-induced hepatitis. These results reveal the small-molecule Sib as a new RIPK1 inhibitor potentially of interest for the treatment of immune-dependent hepatitis.

Endogenous IL-33 has no effect on the progression of fibrosis during experimental steatohepatitis.

Interleukin (IL)-33 has been recently reported to be strongly pro-fibrogenic in various models of liver disease. Our aim was to study the role of endogenous IL-33 in a diet-induced model of steatohepatitis. IL-33 deficient mice and wild type (WT) littermates received a high-fat diet (HFD), or a standard diet for 12 weeks. The HFD-induced steatohepatitis was associated with an upregulation of IL-33 transcripts and protein. An insulin tolerance test revealed lower systemic insulin sensitivity in IL-33-/-HFD mice than in WT-HFD mice. Nevertheless, IL-33 deficiency did not affect the severity of liver inflammation by histological and transcriptomic analyses, nor the quantity of liver fibrosis. Livers from HFD mice had more myeloid populations, markedly fewer NKT cells and higher proportion of ST2+ Treg cells and ST2+ type 2 innate lymphoid cells (ILC2), all unaffected by IL-33 deficiency. In conclusion, deficiency of endogenous IL-33 does not affect the evolution of experimental diet-induced steatohepatitis towards liver fibrosis.

Endogenous IL-33 Deficiency Exacerbates Liver Injury and Increases Hepatic Influx of Neutrophils in Acute Murine Viral Hepatitis.

The alarmin IL-33 has been described to be upregulated in human and murine viral hepatitis. However, the role of endogenous IL-33 in viral hepatitis remains obscure. We aimed to decipher its function by infecting IL-33-deficient mice (IL-33 KO) and their wild-type (WT) littermates with pathogenic mouse hepatitis virus (L2-MHV3). The IL-33 KO mice were more sensitive to L2-MHV3 infection exhibiting higher levels of AST/ALT, higher tissue damage, significant weight loss, and earlier death. An increased depletion of B and T lymphocytes, NKT cells, dendritic cells, and macrophages was observed 48 h postinfection (PI) in IL-33 KO mice than that in WT mice. In contrast, a massive influx of neutrophils was observed in IL-33 KO mice at 48 h PI. A transcriptomic study of inflammatory and cell-signaling genes revealed the overexpression of IL-6, TNFα, and several chemokines involved in recruitment/activation of neutrophils (CXCL2, CXCL5, CCL2, and CCL6) at 72 h PI in IL-33 KO mice. However, the IFNγ was strongly induced in WT mice with less profound expression in IL-33 KO mice demonstrating that endogenous IL-33 regulated IFNγ expression during L2-MHV3 hepatitis. In conclusion, we demonstrated that endogenous IL-33 had multifaceted immunoregulatory effect during viral hepatitis via induction of IFNγ, survival effect on immune cells, and infiltration of neutrophils in the liver.

RIPK1 protects hepatocytes from Kupffer cells-mediated TNF-induced apoptosis in mouse models of PAMP-induced hepatitis.

BACKGROUND & AIMS: The severity of liver diseases is exacerbated by the death of hepatocytes, which can be induced by the sensing of pathogen associated molecular patterns (PAMPs) derived from the gut microbiota. The molecular mechanisms regulating these cell death pathways are poorly documented. In this study, we investigated the role of the receptor interacting protein kinase 1 (RIPK1), a protein known to regulate cell fate decisions, in the death of hepatocytes using two in vivo models of PAMP-induced hepatitis. METHODS: Hepatitis was induced in mice by independent injections of two different bacterial PAMPs: lipopolysaccharide (LPS) and unmethylated CpG oligodeoxynucleotide (CpG-DNA) motifs. The role of RIPK1 was evaluated by using mice specifically lacking RIPK1 in liver parenchymal cells (Ripk1LPC-KO). Administration of liposome-encapsulated clodronate served to investigate the role of Kupffer cells in the establishment of the disease. Etanercept, a tumor necrosis factor (TNF)-decoy receptor, was used to study the contribution of TNF-α during LPS-mediated liver injury. RESULTS: Whereas RIPK1 deficiency in liver parenchymal cells did not trigger basal hepatolysis, it greatly sensitized hepatocytes to apoptosis and liver damage following a single injection of LPS or CpG-DNA. Importantly, hepatocyte death was prevented by previous macrophage depletion or by TNF inhibition. CONCLUSIONS: Our data highlight the pivotal function of RIPK1 in maintaining liver homeostasis in conditions of macrophage-induced TNF burst in response to PAMPs sensing. LAY SUMMARY: Excessive death of hepatocytes is a characteristic of liver injury. A new programmed cell death pathway has been described involving upstream death ligands such as TNF and downstream kinases such as RIPK1. Here, we show that in the presence of LPS liver induced hepatic injury was due to secretion of TNF by liver macrophages, and that RIPK1 acts as a powerful protector of hepatocyte death. This newly identified pathway in the liver may be helpful in the management of patients to predict their risk of developing acute liver failure.

RIPK1 protects from TNF-α-mediated liver damage during hepatitis.

Cell death of hepatocytes is a prominent characteristic in the pathogenesis of liver disease, while hepatolysis is a starting point of inflammation in hepatitis and loss of hepatic function. However, the precise molecular mechanisms of hepatocyte cell death, the role of the cytokines of hepatic microenvironment and the involvement of intracellular kinases, remain unclear. Tumor necrosis factor alpha (TNF-α) is a key cytokine involved in cell death or survival pathways and the role of RIPK1 has been associated to the TNF-α-dependent signaling pathway. We took advantage of two different deficient mouse lines, the RIPK1 kinase dead knock-in mice (Ripk1K45A) and the conditional knockout mice lacking RIPK1 only in liver parenchymal cells (Ripk1LPC-KO), to characterize the role of RIPK1 and TNF-α in hepatitis induced by concanavalin A (ConA). Our results show that RIPK1 is dispensable for liver homeostasis under steady-state conditions but in contrast, RIPK1 kinase activity contributes to caspase-independent cell death induction following ConA injection and RIPK1 also serves as a scaffold, protecting hepatocytes from massive apoptotic cell death in this model. In the Ripk1LPC-KO mice challenged with ConA, TNF-α triggers apoptosis, responsible for the observed severe hepatitis. Mechanism potentially involves both TNF-independent canonical NF-κB activation, as well as TNF-dependent, but canonical NF-κB-independent mechanisms. In conclusion, our results suggest that RIPK1 kinase activity is a pertinent therapeutic target to protect liver against excessive cell death in liver diseases.

Toll-like receptor-2 exacerbates murine acute viral hepatitis.

Viral replication in the liver is generally detected by cellular endosomal Toll-like receptors (TLRs) and cytosolic helicase sensors that trigger antiviral inflammatory responses. Recent evidence suggests that surface TLR2 may also contribute to viral detection through recognition of viral coat proteins but its role in the outcome of acute viral infection remains elusive. In this study, we examined in vivo the role of TLR2 in acute infections induced by the highly hepatotrophic mouse hepatitis virus (MHV) type 3 and weakly hepatotrophic MHV-A59 serotype. To address this, C57BL/6 (wild-type; WT) and TLR2 knockout (KO) groups of mice were intraperitoneally infected with MHV3 or MHV-A59. MHV3 infection provoked a fulminant hepatitis in WT mice, characterized by early mortality and high alanine and aspartate transaminase levels, histopathological lesions and viral replication whereas infection of TLR2 KO mice was markedly less severe. MHV-A59 provoked a comparable mild and subclinical hepatitis in WT and TLR2 KO mice. MHV3-induced fulminant hepatitis in WT mice correlated with higher hepatic expression of interferon-ß, interleukin-6, tumour necrosis factor-α, CXCL1, CCL2, CXCL10 and alarmin (interleukin-33) than in MHV-A59-infected WT mice and in MHV3-infected TLR2 KO mice. Intrahepatic recruited neutrophils, natural killer cells, natural killer T cells or macrophages rapidly decreased in MHV3-infected WT mice whereas they were sustained in MHV-A59-infected WT mice and MHV3-infected TLR2 KO. MHV3 in vitro infection of macrophagic cells induced rapid and higher viral replication and/or interleukin-6 induction in comparison to MHV-A59, and depended on viral activation of TLR2 and p38 mitogen-activated protein kinase. Taken together, these results support a new aggravating inflammatory role for TLR2 in MHV3-induced acute fulminant hepatitis.

Ablation of interaction between IL-33 and ST2+ regulatory T cells increases immune cell-mediated hepatitis and activated NK cell liver infiltration.

The IL-33/ST2 axis plays a protective role in T-cell-mediated hepatitis, but little is known about the functional impact of endogenous IL-33 on liver immunopathology. We used IL-33-deficient mice to investigate the functional effect of endogenous IL-33 in concanavalin A (Con A)-hepatitis. IL-33(-/-) mice displayed more severe Con A liver injury than wild-type (WT) mice, consistent with a hepatoprotective effect of IL-33. The more severe hepatic injury in IL-33(-/-) mice was associated with significantly higher levels of TNF-α and IL-1ß and a larger number of NK cells infiltrating the liver. The expression of Th2 cytokines (IL-4, IL-10) and IL-17 was not significantly varied between WT and IL-33(-/-) mice following Con A-hepatitis. The percentage of CD25(+) NK cells was significantly higher in the livers of IL-33(-/-) mice than in WT mice in association with upregulated expression of CXCR3 in the liver. Regulatory T cells (Treg cells) strongly infiltrated the liver in both WT and IL-33(-/-) mice, but Con A treatment increased their membrane expression of ST2 and CD25 only in WT mice. In vitro, IL-33 had a significant survival effect, increasing the total number of splenocytes, including B cells, CD4(+) and CD8(+) T cells, and the frequency of ST2(+) Treg cells. In conclusion, IL-33 acts as a potent immune modulator protecting the liver through activation of ST2(+) Treg cells and control of NK cells.

Potential Therapeutic Aspects of Alarmin Cytokine Interleukin 33 or Its Inhibitors in Various Diseases.

PURPOSE: The purpose of this review was to examine the comprehensively accumulated data regarding potential therapeutic aspects of exogenous administration of interleukin 33 (IL-33) or its antagonists in allergic, cancerous, infectious, and inflammatory diseases. METHODS: A selected review was undertaken of publications that examined the protective and exacerbating effects of IL-33 or its inhibitors in different diseases. Mechanisms of action are summarized to examine the putative role of IL-33 in various diseases. FINDINGS: IL-33 promoted antibacterial, antiviral, anti-inflammatory, and vaccine adjuvant functions. However, in TH2-biased respiratory, allergic, parasitic, and inflammatory conditions, IL-33 exhibited disease-sensitizing effects. The alarmin cytokine IL-33 induced protective effects in diseases via recruitment of regulatory T cells; antiviral CD8(+) cells, natural killer cells, γδ T cells, and nuocytes; antibacterial and antifungal neutrophils or macrophages; vaccine-associated B/T cells; and inhibition of nuclear factor-κB-mediated gene transcription. In contrast, IL-33 exacerbated the disease process by increasing TH2 cytokines, IgE and eosinophilic immune responses, and inhibition of leukocyte recruitment in various diseases. IMPLICATIONS: The protective or exacerbated aspects of use of IL-33 or its inhibitors are dependent on the type of infection or inflammatory condition, duration of disease (acute or chronic), organ involved, cytokine microenvironment, dose or kinetics of IL-33, and genetic predisposition. The alarmin cytokine IL-33 acts at cellular, molecular, and transcriptional levels to mediate pluripotent functions in various diseases and has potential therapeutic value to mitigate the disease process.