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Genetic deletion or pharmacologic inhibition of the Nlrp3 Inflammasome did not ameliorate experimental NASH

Open AccessPublished:January 11, 2023DOI:https://doi.org/10.1016/j.jlr.2023.100330

      Abstract

      It has been postulated that inflammasomes, in particular the NLRP3 inflammasome, mediate the necroinflammation and fibrosis that characterize nonalcoholic steatohepatitis (NASH) by engaging innate immune responses. We aimed to investigate the impact of genetic deletion or pharmacologic inhibition of the NLRP3 inflammasome on experimental steatohepatitis. Global Nlrp3 knockout (expected to inhibit the NLRP3 inflammasome) or Casp1 knockout (expected to inhibit all inflammasomes) mice were compared to wild-type controls after six months on a high-fat, high-cholesterol (HFHC, 1% cholesterol) diet known to induce fibrosing steatohepatitis. Additionally, wild-type mice on a HFHC diet (0.75% or 0.5% cholesterol) for six months were either treated or not treated with an oral, pharmacologic inhibitor of Nlrp3 (MCC950) that was delivered in the drinking water (0.3mg/mL). We found that genetic deletion or pharmacologic inhibition of the NLRP3 inflammasome did not ameliorate any of the histological components of fibrosing NASH in HFHC-fed mice. Collectively, these results do not support NLRP3 inhibition as a potential target for human NASH.

      Keywords

      Abbreviations:

      AST (Aspartate aminotransferase), ALT (Alanine aminotransferase), HOMA-IR (Homeostasis model assessment insulin resistance), LDLR (low-density lipoprotein receptor), NAFLD (nonalcoholic fatty liver disease), NASH (nonalcoholic steatohepatitis), NLRP3 (NLR family pyrin domain containing 3)

      Introduction

      Inflammasomes are cytosolic pattern recognition receptors that recognize “exogenous” pathogen-associated molecular patterns (PAMPs) derived from invading pathogens and “endogenous” danger-associated molecular patterns (DAMPs) derived from damaged tissues. Upon activation by their specific PAMPs and DAMPs, inflammasomes form a multiprotein complex containing a receptor protein, an adaptor and an effector, which induces autocleavage of procaspase -1 into active caspase-1 resulting in the maturation and secretion of IL-1b and IL-18 to engage innate immune responses. Through caspase-1 activation, inflammasome activation can also result in pyroptosis, a form of programmed cell death.
      The NLRP3 inflammasome is one of the best-characterized pattern recognition receptors and is well known for its important role in the regulation of immunity, and the development and progression of various inflammatory diseases. It has been postulated that the NLRP3 inflammasome is also a key sensor of PAMPs/DAMPs in NAFLD and a trigger for necroinflammation and fibrosis, resulting in progression to fibrosing NASH[
      • Seok J.K.
      • Kang H.C.
      • Cho Y.Y.
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      Therapeutic regulation of the NLRP3 inflammasome in chronic inflammatory diseases.
      ]. NASH is characterized by lipotoxicity which could result in release of DAMPs from injured hepatocytes, while leaky gut and microbiome dysregulation could result in PAMPs, including lipopolysaccharide, potentially triggering NLRP3 activation. The NLRP3 inflammasome is highly expressed in liver cells involved in the innate immune response, such as the Kupffer Cells (KCs), but also in hepatocytes. Some prior studies suggested a role for the NLRP3 inflammasome in triggering the necroinflammation that characterizes experimental steatohepatitis in different animal models such as mice fed a methionine-choline deficient (MCD) diet, hyperphagic mice (such the foz/foz mice) and LDLR deficient mice on a high-fat diet[
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      Pharmacologic inhibition of Nlrp3 has been reported to have positive results in ameliorating experimental steatohepatitis in foz/foz mice on an atherogenic diet[
      • Mridha A.R.
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      ] or ApoE-/- mice on a methionine/choline deficient (MCD) diet[
      • Torres S.
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      ]. Constitutive activation of the NLRP3 inflammasome causes hepatic inflammation and fibrosis[
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      ]. Based on these results, therapeutic targeting of NLRP3 as a treatment of patients with NASH has also been considered, either directly targeting the NLRP3 complex with pharmacologic agents or downstream products including IL-1b and IL-18. Pharmacologic inhibitors of NLRP3, some of which are orally bioavailable, have been recently reviewed[
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      ], MCC950[
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      NLRP3 inflammasome blockade reduces liver inflammation and fibrosis in experimental NASH in mice.
      ,
      • Coll R.C.
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      ,
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      ], CY-09[
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      NLRP3 inflammasome inhibitor CY-09 reduces hepatic steatosis in experimental NAFLD mice.
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      ] and IFM-514[
      • Torres S.
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      • Klein S.
      • et al.
      The Specific NLRP3 Antagonist IFM-514 Decreases Fibrosis and Inflammation in Experimental Murine Non-Alcoholic Steatohepatitis.
      ].
      We previously demonstrated that C57BL/6J mice on a high-fat, high-cholesterol (HFHC) diet recapitulate the metabolic phenotype (obesity, insulin resistance), laboratory abnormalities and histological features (fibrosing steatohepatitis with perisinusoidal “chicken-wire” fibrosis) that characterize human NASH[
      • Savard C.
      • Tartaglione E.V.
      • Kuver R.
      • Haigh W.G.
      • Farrell G.C.
      • Subramanian S.
      • et al.
      Synergistic interaction of dietary cholesterol and dietary fat in inducing experimental steatohepatitis.
      ,
      • Ioannou G.N.
      • Haigh W.G.
      • Thorning D.
      • Savard C.
      Hepatic cholesterol crystals and crown-like structures distinguish NASH from simple steatosis.
      ,
      • Ioannou G.N.
      • Subramanian S.
      • Chait A.
      • Haigh W.G.
      • Yeh M.M.
      • Farrell G.C.
      • et al.
      Cholesterol crystallization within hepatocyte lipid droplets and its role in murine NASH.
      ]. Furthermore, mice on a HFHC diet demonstrate hepatic necroinflammation and profound hepatic cholesterol loading and cholesterol crystals presenting a plethora of potential PAMPs and DAMPs for NLRP3 activation. Therefore, we investigated whether genetic deletion (using Nlrp3 KO or Casp1 KO mice) or pharmacologic inhibition (using MCC950 a novel oral inhibitor[
      • Mridha A.R.
      • Wree A.
      • Robertson A.A.B.
      • Yeh M.M.
      • Johnson C.D.
      • Van Rooyen D.M.
      • et al.
      NLRP3 inflammasome blockade reduces liver inflammation and fibrosis in experimental NASH in mice.
      ,
      • Coll R.C.
      • Robertson A.A.
      • Chae J.J.
      • Higgins S.C.
      • Munoz-Planillo R.
      • Inserra M.C.
      • et al.
      A small-molecule inhibitor of the NLRP3 inflammasome for the treatment of inflammatory diseases.
      ,
      • Gordon R.
      • Albornoz E.A.
      • Christie D.C.
      • Langley M.R.
      • Kumar V.
      • Mantovani S.
      • et al.
      Inflammasome inhibition prevents alpha-synuclein pathology and dopaminergic neurodegeneration in mice.
      ]) of the NLRP3 inflammasome would result in amelioration of histological steatohepatitis in mice fed a HFHC diet. Our results demonstrate little or no impact of genetic deletion or pharmacologic inhibition of Nlrp3 on NASH in this animal model and call for a more guarded examination of whether NLRP3 targeting would be a viable therapeutic strategy in human NASH.

      METHODS

      Genetic Inhibition of Nlrp3 and Caspase-1

      We employed male mice with a global genetic knockout of either the Nlrp3 gene, targeting only the NLRP3 inflammasome, or the Caspase-1 gene (Casp1), which is the final common pathway for all inflammasomes and therefore results in inhibition of all inflammasomes (Figure 1).
      Figure thumbnail gr1
      Figure 1Schematic representation of inflammasome pathways and rationale for the experimental design. Pharmacologic inhibition with MCC950 blocks the Nlrp3 inflammasome. Genetic deletion of Nlrp3 in the Nlrp3 KO mice blocks the Nlrp3 inflammasome while genetic deletion of Casp1 in Casp1 KO mice blocks all the inflammasomes, since activation of Casp1 is the final common pathway for all inflammasomes. We found that neither pharmacologic inhibition of Nlrp3 with MCC950 nor genetic deletion of Nlrp3 or Casp1 ameliorated any of the histological features of fibrosing NASH in high-fat, high-cholesterol fed mice.
      B6N;129-Nlrp3tm1Hhf mice, with a targeted knock-in of a floxed neomycin cassette into intron 2 of the Nlrp3 gene, effectively producing a Nlrp3 expression knockout (Nlrp3 KO) model, and appropriate wild-type controls of the same strain (C57BL/6N) were obtained from the Jackson Laboratory (Bar Harbor, ME). In addition, B6N.129S2-Casp1tm1Flvmice, a Casp1 KO mouse strain, were also obtained from Jackson Laboratory. Wild-type controls for the Casp1 KO mice were the same mice used for the Nlrp3 KO experiment. Starting at 6 months of age, the control and KO mice were given a high-fat (15%, w/w) diet for 6 months supplemented with 1.0% dietary cholesterol purchased from Bio-Serv (Frenchtown, NJ), which has been shown to induce fibrosing steatohepatitis in wild-type mice (3 groups, n=12 mice/group). Cocoa butter, which contains approximately 60% saturated fat, was the source of the extra fat in these diets[
      • Ioannou G.N.
      • Haigh W.G.
      • Thorning D.
      • Savard C.
      Hepatic cholesterol crystals and crown-like structures distinguish NASH from simple steatosis.
      ,
      • Ioannou G.N.
      • Van Rooyen D.M.
      • Savard C.
      • Haigh W.G.
      • Yeh M.M.
      • Teoh N.C.
      • et al.
      Cholesterol-lowering drugs cause dissolution of cholesterol crystals and disperse Kupffer cell crown-like structures during resolution of NASH.
      ]. Their composition is shown in Supplemental Table 1.

      Pharmacologic Inhibition of Nlrp3 Inflammasome

      Starting at 6 months of age, C57BL/6J male mice (Jackson Laboratory, Bar Harbor, ME) were placed on high-fat (15%, w/w) diet supplemented with either 0.75% (24 mice) or 0.5% (18 mice) dietary cholesterol for 6 months. Lower levels of dietary cholesterol (0.75% and 0.5%) were used in this pharmacologic inhibition experiments than in the genetic inhibition experiments (1.0%), because we hypothesized that pharmacologic inhibition might be more likely to ameliorate less severe NASH that occurred at lower dietary cholesterol concentrations - we previously demonstrated a “dose-response” effect of dietary cholesterol[
      • Ioannou G.N.
      • Subramanian S.
      • Chait A.
      • Haigh W.G.
      • Yeh M.M.
      • Farrell G.C.
      • et al.
      Cholesterol crystallization within hepatocyte lipid droplets and its role in murine NASH.
      ]. During the same 6-month period, half the mice on each diet were given access to MCC950 (an orally available NLRP3-specific inflammasome inhibitor) that was dissolved in the water provided ad libitum to each cage at a concentration of 0.3 mg/mL[
      • Gordon R.
      • Albornoz E.A.
      • Christie D.C.
      • Langley M.R.
      • Kumar V.
      • Mantovani S.
      • et al.
      Inflammasome inhibition prevents alpha-synuclein pathology and dopaminergic neurodegeneration in mice.
      ]. Dosing of MCC950 via drinking water at this concentration in C57BL/6J mice was shown to be similarly effective in delivering MCC950 into the blood stream[
      • Gordon R.
      • Albornoz E.A.
      • Christie D.C.
      • Langley M.R.
      • Kumar V.
      • Mantovani S.
      • et al.
      Inflammasome inhibition prevents alpha-synuclein pathology and dopaminergic neurodegeneration in mice.
      ] as when administered by oral gavage[
      • Khan N.
      • Kuo A.
      • Brockman D.A.
      • Cooper M.A.
      • Smith M.T.
      Pharmacological inhibition of the NLRP3 inflammasome as a potential target for multiple sclerosis induced central neuropathic pain.
      ] or intraperitoneal injection[
      • Coll R.C.
      • Robertson A.A.
      • Chae J.J.
      • Higgins S.C.
      • Munoz-Planillo R.
      • Inserra M.C.
      • et al.
      A small-molecule inhibitor of the NLRP3 inflammasome for the treatment of inflammatory diseases.
      ,
      • Primiano M.J.
      • Lefker B.A.
      • Bowman M.R.
      • Bree A.G.
      • Hubeau C.
      • Bonin P.D.
      • et al.
      Efficacy and Pharmacology of the NLRP3 Inflammasome Inhibitor CP-456,773 (CRID3) in Murine Models of Dermal and Pulmonary Inflammation.
      ,
      • Marin-Aguilar F.
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      • Alcocer-Gomez E.
      • Lendines-Cordero D.
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      NLRP3 Inflammasome Inhibition by MCC950 in Aged Mice Improves Health via Enhanced Autophagy and PPARalpha Activity.
      ,
      • Qu J.
      • Yuan Z.
      • Wang G.
      • Wang X.
      • Li K.
      The selective NLRP3 inflammasome inhibitor MCC950 alleviates cholestatic liver injury and fibrosis in mice.
      ]. Fresh diluted MCC950 was added to the water bottles twice per week (changed after 3 or 4 days) for each cage while the control mice received untreated sterile water. Once a month, the water consumption (g fluid/mouse/day respectively) was calculated by measuring the difference in the weight of the water added in the water bottle and the weight of the water remaining four days later. This procedure was immediately repeated over another three-day period. The actual dose of MCC950 ingested by the mice was calculated from the water consumption results (g/mouse/day) times the MCC950 concentration of the water (0.3mg/ml). There was no difference in the water intake of the mice given MCC950 versus untreated water. Based on the daily intake of MCC950 (g/mouse/day) and the body weight of the mice at each time point, we calculated that the mice ingested an MCC950 dose of 19.1-26.7 mg per Kg/day. This dose is comparable to the MCC950 dose administered in other experiments using gavage or intraperitoneal injection.

      Animal procedures

      Mice were housed up to 4 per cage with unrestricted access to food and water. Mice underwent phlebotomy and were euthanized 6 months after initiation of the experimental diets by cervical dislocation following isoflurane anesthesia, and their livers were harvested as well as subcutaneous and intra-abdominal (epididymal) adipose tissue. All experimental procedures were approved by the Institutional Animal Care and Use Committee of the Veterans Affairs Puget Sound Health Care System.

      Histological assessment of hepatic steatosis, inflammation and fibrosis and cholesterol crystals

      Formalin-fixed, paraffin-embedded liver tissue sections were stained with hematoxylin and eosin (H&E), Masson’s trichrome or Sirius red (for collagen). Histological steatosis, inflammation, and fibrosis were assessed semi-quantitatively using the scoring system of Kleiner et al.[
      • Kleiner D.E.
      • Brunt E.M.
      • Van Natta M.
      • Behling C.
      • Contos M.J.
      • Cummings O.W.
      • et al.
      Design and validation of a histological scoring system for nonalcoholic fatty liver disease.
      ] by consensus of two “blinded”, expert hepatopathologists (IS and MY). Sirius red-stained collagen fibers were also quantified using a polarizing microscope by digital image analysis (NIH Image J density software[
      • Savard C.
      • Tartaglione E.V.
      • Kuver R.
      • Haigh W.G.
      • Farrell G.C.
      • Subramanian S.
      • et al.
      Synergistic interaction of dietary cholesterol and dietary fat in inducing experimental steatohepatitis.
      ]), as the average of 12 random 200x-fields without major blood vessels. Hepatic cholesterol crystals were visualized as we previously described[
      • Ioannou G.N.
      • Haigh W.G.
      • Thorning D.
      • Savard C.
      Hepatic cholesterol crystals and crown-like structures distinguish NASH from simple steatosis.
      ,
      • Ioannou G.N.
      • Subramanian S.
      • Chait A.
      • Haigh W.G.
      • Yeh M.M.
      • Farrell G.C.
      • et al.
      Cholesterol crystallization within hepatocyte lipid droplets and its role in murine NASH.
      ,
      • Ioannou G.N.
      • Van Rooyen D.M.
      • Savard C.
      • Haigh W.G.
      • Yeh M.M.
      • Teoh N.C.
      • et al.
      Cholesterol-lowering drugs cause dissolution of cholesterol crystals and disperse Kupffer cell crown-like structures during resolution of NASH.
      ,
      • Ioannou G.N.
      • Lee S.P.
      • Linsley P.S.
      • Gersuk V.
      • Yeh M.M.
      • Chen Y.Y.
      • et al.
      Pcsk9 Deletion Promotes Murine Nonalcoholic Steatohepatitis and Hepatic Carcinogenesis: Role of Cholesterol.
      ,
      • Ioannou G.N.
      • Landis C.S.
      • Jin G.Y.
      • Haigh W.G.
      • Farrell G.C.
      • Kuver R.
      • et al.
      Cholesterol Crystals in Hepatocyte Lipid Droplets Are Strongly Associated With Human Nonalcoholic Steatohepatitis.
      ] by observing fresh-frozen liver sections under polarized light.

      Hepatic and adipose tissue lipid analysis

      Lipids were extracted from frozen mouse liver or adipose tissue (subcutaneous and intra-abdominal) by using the Folch method[
      • Folch J.
      • Lees M.
      • Sloane Stanley G.H.
      A simple method for the isolation and purification of total lipides from animal tissues.
      ]. The neutral lipid fractions were prepared by solid phase extraction, and the triglycerides, cholesterol esters, and free cholesterol were then separated and quantified by normal phase HPLC/Evaporative Light Scattering Detector (ELSD).

      Other measurements

      Blood specimens were collected immediately prior to sacrifice after a 4-hour fast and tested for plasma cholesterol, triglycerides, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), glucose, insulin and high molecular weight (HMW) adiponectin. Average food consumption was measured monthly.
      To confirm the knock-out of Nlrp3 in the B6N;129-Nlrp3tm1Hhf mice, we measured mRNA expression of the Nlrp3 gene in liver tissue. Western blot analysis was used to measure protein levels of both the inactive pro-caspase-1 and active caspase-1 in liver tissue to confirm Casp1 deletion in B6N.129S2-Casp1tm1Flvmice and to confirm adequate inhibition of the Nlrp3 inflammasome by MCC950. We also measured levels of activated Il-1b in plasma using a multiplex assay (Enhanced sensitivity Cytometric Bead Array system, BD Biosciences, Franklin Lakes NJ).

      Results

      Genetic deletion of Nlrp3 and Casp1 did not lead to amelioration of fibrosing NASH

      The wild-type mice developed obesity, hyperglycemia, elevated serum liver enzymes and hepatic steatosis, inflammation and perisinusoidal fibrosis characteristic of fibrosing NASH, after 6 months on the high fat/ 1% cholesterol diet (Table 1 and Figure 2), as we previously described[
      • Savard C.
      • Tartaglione E.V.
      • Kuver R.
      • Haigh W.G.
      • Farrell G.C.
      • Subramanian S.
      • et al.
      Synergistic interaction of dietary cholesterol and dietary fat in inducing experimental steatohepatitis.
      ,
      • Ioannou G.N.
      • Haigh W.G.
      • Thorning D.
      • Savard C.
      Hepatic cholesterol crystals and crown-like structures distinguish NASH from simple steatosis.
      ,
      • Ioannou G.N.
      • Subramanian S.
      • Chait A.
      • Haigh W.G.
      • Yeh M.M.
      • Farrell G.C.
      • et al.
      Cholesterol crystallization within hepatocyte lipid droplets and its role in murine NASH.
      ].
      Table 1Comparison of Nlrp3 KO and Casp1 KO mice with wild-type mice after 6 months on a high-fat/high-cholesterol (HFHC) diet
      Genetic StrainWild-typeNlrp3 KOCasp1 KO
      Genetic backgroundC57BL/6NC57BL/6NC57BL/6N
      DietHFHC

      1.0% Cholesterol
      HFHC

      1.0% Cholesterol
      HFHC

      1.0% Cholesterol
      Number of mice121010
      Body Weight (g)46.2 ± 1.950.6 ± 4.4
      t-test p<0.05 compared to wild-type
      45.6 ± 2.9
      Liver Weight (g)4.3 ± 0.45.3 ± 1.0
      t-test p<0.05 compared to wild-type
      3.8 ± 0.5
      t-test p<0.05 compared to wild-type
      Liver Weight/Body Weight (%)9.4 ± 0.710.5 ± 1.2
      t-test p<0.05 compared to wild-type
      8.2 ± 0.7
      t-test p<0.01 compared to wild-type
      Food Consumption (g/mouse/day)2.9 ± 0.13.3 ± 0.2
      t-test p<0.01 compared to wild-type
      2.5 ± 0.2
      t-test p<0.01 compared to wild-type
      Plasma Levels (fasting)
      ALT (U/L)382 ± 104.5424.7 ± 163.1391.6 ± 142.1
      AST (U/L)276.9 ± 78.4309.0 ± 87.1291.6 ± 117.2
      ALP (U/L)105.3 ± 17.9147.0 ± 43.2
      t-test p<0.05 compared to wild-type
      104.9 ± 25.9
      Cholesterol (mg/dL)303.1 ± 40.7344.6 ± 59.0328.4 ± 56.3
      Triglyceride (mg/dL)62.9 ± 12.769.3 ± 10.869.3 ± 13.3
      Glucose (mg/dL)348.1 ± 84.6316.4 ± 39.0377.0 ± 38.1
      Insulin (ng/ml)2.01 ± 1.334.93 ± 3.921.76 ± 1.22
      HOMA-IR37.9 ± 25.582.2 ± 62.533.9 ± 22.3
      HMW-Adiponectin (mg/ml)1.49 ± 0.341.61 ± 0.672.16 ± 0.23
      t-test p<0.05 compared to wild-type
      Hepatic Histology†, n (%)
      Steatosis grade
      00 (0%)0 (0%)0 (0%)
      10 (0%)0 (0%)0 (0%)
      20 (0%)0 (0%)0 (0%)
      312 (100%)10 (100%)10 (100%)
      Inflammation grade
      00 (0%)0 (0%)0 (0%)
      10 (0%)0 (0%)1 (10%)
      211 (92%)6 (60%)6 (60%)
      31 (8%)4 (40%)3 (30%)
      Fibrosis stage
      02 (17%)0 (0%)5 (50%)
      110 (83%)9 (90%)5 (50%)
      20 (0%)1 (10%)0 (0%)
      30 (0%)0 (0%)0 (0%)
      Sirius Red Staining (fibrosis)B, % area1.00 ± 0.642.00 ± 1.921.19 ± 0.70
      Hepatic Cholesterol BirefringenceVery CommonVery CommonVery Common
      Hepatic Lipids (mg/ g liver)
      Cholesterol Ester78.5 ± 32.6122.9 ± 42.3
      t-test p<0.01 compared to wild-type
      106.2 ± 28.0
      t-test p<0.05 compared to wild-type
      Free Cholesterol0.52 ± 0.470.62 ± 0.311.30 ± 0.45
      t-test p<0.01 compared to wild-type
      Triacylglycrides345.5 ± 72.2295.2 ± 69.8426.3 ± 67.3
      t-test p<0.05 compared to wild-type
      Diacylglycerides1.34 ± 0.271.46 ± 0.31.41 ± 0.25
      Epididymal Fat (mg/ g fat)
      Cholesterol Ester0.52 ± 0.220.43 ± 0.140.39 ± 0.13
      Free Cholesterol3.19 ± 1.123.48 ± 0.912.03 ± 0.44
      t-test p<0.05 compared to wild-type
      Subcutaneous Fat (mg/ g fat)
      Cholesterol Ester0.23 ± 0.090.20 ± 0.090.16 ± 0.06
      Free Cholesterol1.64 ± 0.831.51 ± 0.391.42 ± 0.34
      t-test p<0.01 compared to wild-type
      ∗∗ t-test p<0.05 compared to wild-type
      Figure thumbnail gr2
      Figure 2Comparison of hepatic histological features in wild-type mice versus Nlrp3 KO and Casp1 KO mice after 6 months on a HFHC (1% cholesterol) diet. The wild type mice developed similar grades of steatosis (top row), perisinusoidal fibrosis (middle row) and hepatocyte lipid droplet cholesterol crystallization (lower row) as the Nlrp3 KO and Casp1 KO mice.
      The Nlrp3 KO mice on the same diet had slightly higher body weight, liver weight, liver weight/body weight ratio, and average food consumption. The Nlrp3 KO mice also developed fibrosing NASH with similar elevated levels of plasma ALT, AST, and ALP compared to the wild-type mice, similar levels of hepatic steatosis, and slightly higher levels of hepatic inflammation grade and hepatic fibrosis by histological staging and quantitative Sirius red staining – a difference that was not statistically significant (Table 1). Hepatic levels of cholesterol ester were higher in the Nlrp3 KO mice than wild-type mice, with no difference in hepatic free cholesterol or triacylglyceride levels. Hepatic cholesterol crystals were equally prominent in frozen liver sections of both Nlrp3 KO and wild-type mice (Table 1 and Figure 2).
      The free cholesterol and cholesterol ester contents of the epididymal and subcutaneous fat were not different between Nlrp3 KO and wild-type mice (Table 1).
      The Casp1 KO mice also developed fibrosing NASH and compared to wild-type mice, they had similar body weight, liver weight/body weight ratio, average food consumption, plasma levels of ALT, AST and ALP and hepatic steatosis, inflammation and fibrosis measured by Sirius red.

      Pharmacologic Inhibition of NLRP3 Inflammasome with MCC950 did not lead to amelioration of fibrosing NASH

      Mice on a 0.75% cholesterol diet that did not receive MCC950 developed fibrosing NASH, albeit with lower levels of fibrosis by histological staging or quantitative Sirius red staining than the mice treated with 1% cholesterol in the genetic deletion experiments described above. Compared to mice on a 0.75% cholesterol diet that were not treated with MCC950, those that were treated with MCC950 in their drinking water had lower liver weight and plasma cholesterol, similar levels of plasma ALT, AST and ALP, similar levels of hepatic steatosis and inflammation and slightly lower levels of hepatic fibrosis by quantitative Sirius red staining or by histological stage that were not statistically significant (Table 2 and Figure 3).
      Table 2Comparison of MCC950-treated
      MCC950 was dissolved in the drinking water of the mice at a concentration of 0.3mg/ml.
      mice to untreated mice fed a HFHC diet for 6 months
      Genetic backgroundC57BL/6JC57BL/6JC57BL/6JC57BL/6J
      DietHFHC

      0.75% Cholesterol
      HFHC

      0.75% Cholesterol
      HFHC

      0.5% Cholesterol
      HFHC

      0.5% Cholesterol
      MCC950 TreatmentNoYesNoYes
      Number of mice111098
      Body Weight (g)48.7± 4.045.9 ± 4.243.5 ± 5.044.9 ± 7.9
      Liver Weight (g)4.4 ± 0.93.6 ± 0.9
      t-test p<0.05 compared to No MCC950 group
      2.8 ± 1.23.2 ± 1.7
      Liver Weight/Body Weight (%)9.0 ± 1.07.8 ± 1.3
      t-test p<0.05 compared to No MCC950 group
      6.2 ± 2.26.8 ± 2.6
      Food Consumption (g/mouse/day)3.1 ± 0.33.1 ± 0.43.0 ± 0.33.0 ± 0.4
      Fluid Consumption (g/mouse/day)2.8 ± 0.22.9 ± 0.33.0 ± 0.33.1 ± 0.4
      MCC950 Dose (mg/Kg/ day)None19.2 ± 1.9None20.7± 2.9
      Plasma Levels (fasting)
      ALT (U/L)365.3 ± 57.1335.1 ± 122.7171.3 ± 215.9186.0 ± 203.8
      AST (U/L)261.7 ± 44.4269.4 ± 156.1139.3 ± 135.3146.2 ± 128.6
      ALP (U/L)216.9 ± 30.1199.0 ± 53.4126.4 ± 50.1140.5 ± 65.0
      Cholesterol (mg/dL)323.1 ± 38.5232.5 ± 93.3
      t-test p<0.01 compared to No MCC950 group
      196.9 ± 58.1205.2 ± 117.8
      Triglyceride (mg/dL)43.7 ± 9.138.5 ± 9.546.0 ± 13.944.8 ± 19.1
      Glucose (mg/dL)293.9 ± 33.3248.8 ± 44.7
      t-test p<0.05 compared to No MCC950 group
      289.2 ± 49.6304.0 ± 28.2
      Hepatic Histology
      MCC950 was dissolved in the drinking water of the mice at a concentration of 0.3mg/ml.
      , n (%)
      Steatosis grade
      00 (0%)0 (0%)2 (22%)2 (25%)
      10 (0%)0 (0%)0 (0%)0 (0%)
      20 (0%)0 (0%)0 (0%)0 (0%)
      311 (100%)10 (100%)7 (78%)6 (75%)
      Inflammation grade
      00 (0%)0 (0%)0 (0%)0 (0%)
      10 (0%)0 (0%)0 (0%)2 (25%)
      20 (0%)0 (0%)4 (44%)1 (12%)
      311 (100%)10 (100%)5 (56%)5 (63%)
      Fibrosis stage
      04 (36%)7 (70%)2 (22%)/2 (25%)
      17 (64%)3 (30%)7 (78%)5 (63%)
      20 (0%)0 (0%)0 (0%)1 (12%)
      30 (0%)0 (0%)0 (0%)0 (0%)
      Sirius Red Staining (fibrosis)B, % area0.37 ± 0.440.17 ± 0.120.16 ± 0.140.31 ± 0.35
      Hepatic Cholesterol CrystalsVery commonVery commonUncommon/ widely scatteredUncommon/ widely scattered
      Hepatic Lipids (mg/ g liver)
      Cholesterol Ester72.1 ± 13.352.0 ± 14.7
      t-test p<0.01 compared to No MCC950 group
      19.1 ± 15.226.6 ± 31.4
      Free Cholesterol1.17 ± 0.600.78 ± 0.220.57 ± 0.490.45 ± 0.36
      Triacylglycerides108.2 ± 36.2110.4 ± 24.4122.4 ± 42.376.1 ± 48.4
      Diacylglycerides0.35 ± 0.290.27 ± 0.090.15 ± 0.070.21 ± 0.14
      MCC950 was dissolved in the drinking water of the mice at a concentration of 0.3mg/ml.
      t-test p<0.05 compared to No MCC950 group
      ∗∗ t-test p<0.01 compared to No MCC950 group
      Figure thumbnail gr3
      Figure 3Comparison of wild-type mice on HFHC diet (0.75% or 0.5% cholesterol) that were either treated with MCC950 in their drinking water or not. There was little difference in MCC950 treated versus Control mice in the degree of hepatic fibrosis (Sirius Red staining) or cholesterol crystallization in hepatocyte lipid droplets (unstained/polarized light) for both 0.75% and 0.5% cholesterol diets
      Mice fed a high fat diet with 0.5% cholesterol generally had lower levels of serum AST, ALT and ALP and lower levels of steatosis, inflammation, or quantitative fibrosis than mice fed 0.75% cholesterol (Table 2 and Figure 3). Among mice on a 0.5% cholesterol, there were no differences between those that received MCC950 and those that did not in all parameters measured, including levels of plasma AST, ALT and ALP, hepatic lipid composition and hepatic steatosis, inflammation, and fibrosis.

      Confirmation of NLRP3 inflammasome inhibition in our genetic and pharmacologic experimental models

      We confirmed the knock-out of Nlrp3 in the B6N;129-Nlrp3tm1Hhf mice by measuring mRNA expression of the Nlrp3 gene in liver tissue, which showed no detectable mRNA in the KO mouse livers compared to high expression levels in the WT mice.
      We measured pro-caspase-1 and active-caspase-1 protein levels by Western blot in liver tissue (Supplemental Figure 1). Wild-type mice treated with water (Control) in our pharmacologic inhibition experiments demonstrated a band for both pro-caspase-1 and active caspase-1 as expected.
      Wild-type mice treated with MCC950, the pharmacologic inhibitor of Nlrp3, demonstrated a band for pro-caspase-1 but no band for active-caspase-1, confirming that MCC950 truly inhibited the Nrlp3 inflammasome and prevented conversion of pro-caspase-1 to active-caspase-1. Casp1 knockout (KO) mice demonstrated no band for either pro-caspase-1 or active caspase-1, confirming that these mice were true knockouts.
      Plasma Il-1b levels were extremely low with high variability between samples which precluded us from demonstrating any statistically significant differences between the groups, as shown in Supplemental Figure 2, except for the Nlrp3 KO mice, which had significantly lower levels as expected.

      Discussion

      We found that genetic deletion of Nlrp3 or Cas1, or pharmacologic inhibition of Nlrp3 using an oral inhibitor (MCC950) did not ameliorate the development of any of the histological features of fibrosing steatohepatitis (hepatic fibrosis, inflammation, or steatosis) in HFHC-fed mice. In contrast to prior experiments, our results temper the enthusiasm for Nlrp3 inhibition as a therapeutic strategy in human NASH.
      There are no approved pharmacotherapies for NASH. The NLRP3 inflammasome has been suggested as an attractive potential target for NASH therapies for a number of reasons. First, the NLRP3 inflammasome has been shown to be activated in some mouse models of NASH. Second, inflammasome activation results in caspase-1 cleavage and activation, which in turn causes activation of pro-inflammatory mediators IL-1b and IL-18 as well as inducing pyroptosis, all factors thought to be relevant in NASH pathogenesis. Third, candidate PAMPs and DAMPs that can potentially initiate NLRP3 activation are prominent in NASH. Indeed, prior studies in animal models suggested that inhibition of Nlrp3 ameliorated some histologic components of NASH whereas constitutive activation of Nlrp3 exacerbated NASH features[
      • Mridha A.R.
      • Wree A.
      • Robertson A.A.B.
      • Yeh M.M.
      • Johnson C.D.
      • Van Rooyen D.M.
      • et al.
      NLRP3 inflammasome blockade reduces liver inflammation and fibrosis in experimental NASH in mice.
      ,
      • Wree A.
      • Eguchi A.
      • McGeough M.D.
      • Pena C.A.
      • Johnson C.D.
      • Canbay A.
      • et al.
      NLRP3 inflammasome activation results in hepatocyte pyroptosis, liver inflammation, and fibrosis in mice.
      ,
      • Wree A.
      • McGeough M.D.
      • Pena C.A.
      • Schlattjan M.
      • Li H.
      • Inzaugarat M.E.
      • et al.
      NLRP3 inflammasome activation is required for fibrosis development in NAFLD.
      ,
      • Csak T.
      • Pillai A.
      • Ganz M.
      • Lippai D.
      • Petrasek J.
      • Park J.K.
      • et al.
      Both bone marrow-derived and non-bone marrow-derived cells contribute to AIM2 and NLRP3 inflammasome activation in a MyD88-dependent manner in dietary steatohepatitis.
      ,
      • Morrison M.C.
      • Mulder P.
      • Salic K.
      • Verheij J.
      • Liang W.
      • van Duyvenvoorde W.
      • et al.
      Intervention with a caspase-1 inhibitor reduces obesity-associated hyperinsulinemia, non-alcoholic steatohepatitis and hepatic fibrosis in LDLR-/-.Leiden mice.
      ,
      • Torres S.
      • Brol M.J.
      • Magdaleno F.
      • Schierwagen R.
      • Uschner F.E.
      • Klein S.
      • et al.
      The Specific NLRP3 Antagonist IFM-514 Decreases Fibrosis and Inflammation in Experimental Murine Non-Alcoholic Steatohepatitis.
      ,
      • Kaufmann B.
      • Leszczynska A.
      • Reca A.
      • Booshehri L.M.
      • Onyuru J.
      • Tan Z.
      • et al.
      NLRP3 activation in neutrophils induces lethal autoinflammation, liver inflammation, and fibrosis.
      ,
      • Gallego P.
      • Castejon-Vega B.
      • Del Campo J.A.
      • Cordero M.D.
      The Absence of NLRP3-inflammasome Modulates Hepatic Fibrosis Progression, Lipid Metabolism, and Inflammation in KO NLRP3 Mice during Aging.
      ]. Given this promising potential of Nlrp3 inhibition as a treatment for NASH, we undertook a systematic investigation of the effects of both genetic deletion and pharmacologic inhibition of Nlrp3 on experimental steatohepatitis. We used an animal model of NASH, the HFHC-fed mouse, that provides a plethora of potential DAMPs (including cholesterol crystals) known to activate Nlpr3 as well as recapitulating the histologic and metabolic phenotype of chronic NASH. However, we found that neither genetic deletion nor pharmacologic inhibition of Nlrp3 ameliorated any of the histologic features of NASH. Furthermore, in contrast to some prior studies[
      • Morrison M.C.
      • Mulder P.
      • Salic K.
      • Verheij J.
      • Liang W.
      • van Duyvenvoorde W.
      • et al.
      Intervention with a caspase-1 inhibitor reduces obesity-associated hyperinsulinemia, non-alcoholic steatohepatitis and hepatic fibrosis in LDLR-/-.Leiden mice.
      ] [
      • Dixon L.J.
      • Flask C.A.
      • Papouchado B.G.
      • Feldstein A.E.
      • Nagy L.E.
      Caspase-1 as a central regulator of high fat diet-induced non-alcoholic steatohepatitis.
      ], we found that genetic deletion of Casp1, the final common pathway for all inflammasomes, did not result in improved NASH histology. Differences between our findings and prior studies are likely due to differences in the mouse models of NASH that were employed in each study, and only future human studies can resolve whether inhibiting Nlrp3 specifically or all inflammasomes via caspase-1 inhibition can improve human NASH histology – which would also indirectly suggest which experimental design reflects the human condition most closely.
      In a prior study[
      • Mridha A.R.
      • Wree A.
      • Robertson A.A.B.
      • Yeh M.M.
      • Johnson C.D.
      • Van Rooyen D.M.
      • et al.
      NLRP3 inflammasome blockade reduces liver inflammation and fibrosis in experimental NASH in mice.
      ], MCC950 was gavaged daily 5 days/week (20mg/kg/day, in saline) for 8 weeks in female foz/foz mice (which are hyperphagic due to a mutation in the Alms1 gene and develop severe obesity and hyperglycemia) following 16 weeks on an atherogenic diet. In that study, the MCC950-treated mice were less likely to develop steatosis, inflammation or “definite NASH” and had less fibrosis than the untreated mice. It is unclear why MCC950 administration did not result in a similar amelioration of steatosis, inflammation, and fibrosis in our experiments. We administered MCC950 in the drinking water, rather than by gavage, however the average daily dose ingested was almost identical (19.2 and 20.7 mg/Kg/day) to the dose gavaged in the prior study and furthermore in our experiments mice were treated with MCC950 every day (rather than only 5 days/week) and for the entire duration of the HFHC feeding (6 months) rather than only for 8 weeks after the steatohepatitis had already developed in the prior study. The bioavailability of MCC950 administered to mice in the drinking water at 0.3mg/ml and its effectiveness at inhibiting the NLRP3 inflammasome in vivo was established in a prior published study[
      • Gordon R.
      • Albornoz E.A.
      • Christie D.C.
      • Langley M.R.
      • Kumar V.
      • Mantovani S.
      • et al.
      Inflammasome inhibition prevents alpha-synuclein pathology and dopaminergic neurodegeneration in mice.
      ] and this dosage was recommended to us by the manufacturers of MCC950 (personal communication with Dr. Avril Robertson). In another recent study, administration of a different Nlrp3 inhibitor called IFM-514 via intra-peritoneal injection (100mg/kg) daily, 5 days/week for 4 weeks to ApoE-/- mice on a methionine/choline deficient diet was also found to reduce hepatic steatosis, inflammation, and fibrosis[
      • Torres S.
      • Brol M.J.
      • Magdaleno F.
      • Schierwagen R.
      • Uschner F.E.
      • Klein S.
      • et al.
      The Specific NLRP3 Antagonist IFM-514 Decreases Fibrosis and Inflammation in Experimental Murine Non-Alcoholic Steatohepatitis.
      ]. However, when IFM-514 was injected into ApoE-/- mice on a Western diet, it did not reduce inflammation or fibrosis[
      • Torres S.
      • Brol M.J.
      • Magdaleno F.
      • Schierwagen R.
      • Uschner F.E.
      • Klein S.
      • et al.
      The Specific NLRP3 Antagonist IFM-514 Decreases Fibrosis and Inflammation in Experimental Murine Non-Alcoholic Steatohepatitis.
      ]. A different, recently described Nlrp3-specific inhibitor called CY-09 injected daily intraperitoneally in wild-type mice on a high-fat diet for 14 weeks resulted in less weight gain and hepatic steatosis (hepatic inflammation and fibrosis were not reported)[
      • Wang X.
      • Sun K.
      • Zhou Y.
      • Wang H.
      • Zhou Y.
      • Liu S.
      • et al.
      NLRP3 inflammasome inhibitor CY-09 reduces hepatic steatosis in experimental NAFLD mice.
      ]. Although it is impossible to reconcile all these diverse experimental results, we suspect that the type, dose and mode of administration of the Nlrp3 inhibitor and the age, duration, genetic profile, diet and type of lipotoxic injury of the experimental NASH mouse model used all likely affect the observed results. Given conflicting animal model results, it is difficult to predict what the effect of Nlrp3 inhibition in human NASH would be and ultimately clinical trials would be needed to establish this.
      Some noteworthy features of the HFHC-fed mouse model and experimental design that we employed are worth highlighting as they may relate to human NASH. We started our HFHC diets in 6-month-old mice and continued these NASH-inducing diets for another 6 months - as compared to other experimental designs that use much younger mice and shorter interventions. We believe this is more analogous to human, adult NASH which develops most commonly over many years in middle or advanced age. We used non-genetically modified mice for the Nlrp3 inhibition experiments, since human NASH is not driven predominantly or exclusively by a particular genetic polymorphism. C57BL/6J mice on a HFHC diet for at least 6 months develop profound steatosis, necroinflammation and also perisinusoidal fibrosis, which is particularly important because fibrosis is now recognized as the main driver of pathology and adverse clinical outcomes in human NASH and hence treatments for NASH should be targeting fibrosis resolution. On the other hand, C57BL/6J mice, which we used in our MCC950 pharmacologic Nlrp3 inhibition experiments, have a functional defect in NLRP12[
      • Ulland T.K.
      • Jain N.
      • Hornick E.E.
      • Elliott E.I.
      • Clay G.M.
      • Sadler J.J.
      • et al.
      Nlrp12 mutation causes C57BL/6J strain-specific defect in neutrophil recruitment.
      ] which could complicate the interpretation of our results. We also acknowledge that the B6N.129S2-Casp1tm1Flvmice which we employed as a Casp1 knock-out, also has a known loss in caspase-11 gene expression[
      • Kayagaki N.
      • Warming S.
      • Lamkanfi M.
      • Vande Walle L.
      • Louie S.
      • Dong J.
      • et al.
      Non-canonical inflammasome activation targets caspase-11.
      ]. Caspase-11 is involved in a non-canonical inflammasome pathway. However, loss of both caspase 1 and caspase-11 function in B6N.129S2-Casp1tm1Flvmice failed to ameliorate histological NASH.
      The fact that neither Nlrp3 deletion/pharmacologic inhibition nor Casp1 deletion (which blocks the final common pathway for all inflammasomes) ameliorated any of the histologic features of NASH, suggests that at least in the HFHC-fed mouse model these features are not driven predominantly by inflammasome activation. It is possible that other pattern-recognition receptors (PRRs) are involved in sensing DAMPs which are exposed or released upon cell death under “sterile” conditions of lipotoxicity. Such PRR candidates might include transmembrane proteins such as the Toll-like receptors (TLRs)[
      • Arrese M.
      • Cabrera D.
      • Kalergis A.M.
      • Feldstein A.E.
      Innate Immunity and Inflammation in NAFLD/NASH.
      ] and C-type lectin receptors (CLRs, shown to respond to crystalline structures such as uric acid crystals[
      • Neumann K.
      • Castineiras-Vilarino M.
      • Hockendorf U.
      • Hannesschlager N.
      • Lemeer S.
      • Kupka D.
      • et al.
      Clec12a is an inhibitory receptor for uric acid crystals that regulates inflammation in response to cell death.
      ]) as well as cytoplasmic proteins such as Retinoic acid-inducible gene(RIG)-I-like receptors (RLRs)[
      • Takeuchi O.
      • Akira S.
      Pattern recognition receptors and inflammation.
      ].
      In conclusion, although NLRP3 activation can induce hepatic inflammation and fibrosis, inhibiting the NLRP3 pathway does not ameliorate any of the histological features of NASH in an experimental mouse model suggesting that they are not mediated by NLRP3 inflammation.

      Data Availability

      All the data described in the article are located within the article including the supplemental data.

      Conflicts of Interest

      NO CONFLICTS OF INTEREST EXIST.

      Supplementary Data

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