Department of Medicine, University of California, San Diego, School of Medicine, La Jolla, CA
Division of Gastroenterology, Department of Medicine, University of California, San Diego, School of Medicine, 9500 Gilman Drive, MC#0702, Leichtag Biomedical Research Building, Room #118B, La Jolla, CA 92093-0702.
Potential conflict of interest: Nothing to report.
This study was supported by National Institutes of Health Grants R01AA02172 (to E. S.), R01DK085252 (to E. S.), R24DK090962 (to D. A. B.), and R01GM041804 (to D. A. B.); a pilot grant from the University of California, San Diego Digestive Diseases Research Development Center (DK080506) (to E. S.); the Takeda Science Foundation (K. M.); The Mochida Memorial Foundation for Medical and Pharmaceutical Research (K. M.); the Mishima Kaiun Memorial Foundation (K. M.); and a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (to K. M.).
Innate immune signaling associated with Toll-like receptors (TLRs) is a key pathway involved in the progression of nonalcoholic steatohepatitis (NASH). Here we show that both TLR2 and palmitic acid are required for activation of the inflammasome, interleukin (IL)-1α, and IL-1β, resulting in the progression of NASH. Wild-type (WT) and TLR2−/− mice were fed a choline-deficient amino acid–defined (CDAA) diet for 22 weeks to induce NASH. Bone marrow–transplanted TLR2 chimeric mice were generated after the recipient mice were lethally irradiated. Kupffer cells and hepatic stellate cells (HSCs) were isolated from WT mice and stimulated with TLR2 ligand and/or palmitic acid. WT mice on the CDAA diet developed profound steatohepatitis and liver fibrosis. In contrast, TLR2−/− mice had suppressed progression of NASH. Although both Kupffer cells and HSCs respond to TLR2 ligand, TLR2 bone marrow chimeric mice demonstrated that Kupffer cells were relatively more important than HSCs in TLR2-mediated progression of NASH. In vitro, palmitic acid alone did not increase TLR2 signaling-target genes, including cytokines and inflammasome components in Kupffer cells and HSCs. The TLR2 ligand increased Nod-like receptor protein 3, an inflammasome component, in Kupffer cells but not in HSCs. In the presence of TLR2 ligand, palmitic acid did induce caspase-1 activation and release of IL-1α and IL-1β in Kupffer cells; however, these effects were not observed in HSCs. In vivo, WT mice on the CDAA diet showed increased caspase-1 activation in the liver and elevated serum levels of IL-1α and IL-1β levels, which were suppressed in TLR2−/− mice. Conclusion: TLR2 and palmitic acid cooperatively activate the inflammasome in Kupffer cells and/or macrophages in the development of NASH. (HEPATOLOGY 2013;)
Nonalcoholic fatty liver disease (NAFLD), the hepatic consequence of the metabolic syndrome, is a significant public health issue in developed countries. The spectrum of NAFLD ranges from simple steatosis to steatosis with progressive liver inflammation and fibrosis, termed nonalcoholic steatohepatitis (NASH), which eventually causes liver cirrhosis that may increase the prevalence of hepatocellular carcinoma.1,2 However, the majority of mechanisms involved in the multifactorial events in the development of NASH remain unresolved.
The hepatic innate immune system is activated in the pathogenesis of NASH.3,4 Toll-like receptors (TLRs) are pattern recognition signal receptors for sensing bacterial- and viral-derived signature motifs to activate the innate immune system.5 TLR signaling functions in the host defense against invading pathogens through induction of proinflammatory cytokines in immune cells. However, the overactivation of TLR signaling or the breakdown of TLR tolerance produce a large amount of inflammatory cytokines, which ultimately leads to tissue damage.6 Among the 13 TLRs identified in mammals, TLR2, TLR4, and TLR9 are reported to be associated with steatohepatitis.4,7,8 TLR4 and TLR9 have been shown to promote hepatic inflammation and fibrosis in experimental NASH.8,9 However, the role of TLR2 in the pathogenesis of NAFLD is controversial. TLR2 deficiency protected mice from hepatic steatosis induced by a high-fat diet (HFD).10,11 The NASH model induced by a methionine- and choline-deficient (MCD) diet showed that loss of TLR2 increased susceptibility to pathogen-associated steatohepatitis.7,12 These conflicting data imply the complexity of TLR2-mediated NAFLD.
Inflammasome activation is a pathway required for processing interleukin (IL)-1β and IL-18 from their proforms to active forms through cleavage by caspase-1. Recent reports have demonstrated that loss of inflammasome components protects mice from NAFLD induced by a high-fat diet.13,14 In addition, inactivation of IL-1 receptor signaling attenuates mouse models of NASH.9,15 These data suggest that the inflammasome contributes to the development of NAFLD. A two-signal hypothesis is accepted in inflammasome activation: the first signal produces pro–IL-1β and inflammasome components; the second assembles inflammasome components to engage in caspase-1 activation. TLRs, including TLR2, activate the first signals, and the second signals are activated by danger-associated molecular patterns such as extracellular adenosine triphosphate and urate crystals. Emerging evidence has shown that free fatty acids (FFAs) activate the inflammasome16,17 as well as TLRs,18–20 implicating FFAs as promoters of NASH. Elevated plasma FFA concentrations in patients with the metabolic syndrome including NAFLD21 may contribute to the development of NAFLD.
It is often debated which cell types respond to TLR ligands and are responsible for inflammasome activation. Macrophages, including Kupffer cells, activate the inflammasome and produce TLR-induced cytokines. Hepatic nonimmune cells, including hepatic stellate cells (HSCs) and hepatocytes, also respond to TLR ligands and are the site of inflammasome activation.17,22
Here we show that TLR2 is a crucial molecule that promotes liver injury, inflammation, and fibrosis in mice fed a choline-deficient amino acid–defined (CDAA) diet that develop NASH accompanied by obesity and insulin resistance.9,23 TLR2 signaling increased the expression of proinflammatory cytokines, including IL-1β, and the inflammasome component Nod-like receptor protein (NLRP) 3. In cooperation with palmitic acid, the most abundant FFA in the plasma of NAFLD,16,24 TLR2 signaling activates the inflammasome primarily in Kupffer cells, but not in HSCs or hepatocytes.
Wild-type (WT) C57BL/6 mice and TLR2−/− mice purchased from Jackson Laboratories (Bar Harbor, ME) were bred in the University of California, San Diego vivarium. TLR2−/− mice were back-crossed at least 10 generations onto the C57BL/6 background and displayed a similar hepatic phenotype as WT mice under standard laboratory chow. Male mice were divided into two groups at 8 weeks of age: choline-supplemented amino acid–defined (CSAA) diet (catalog# 518754, Dyets Inc. Bethlehem, PA) and CDAA (catalog# 518753, Dyets Inc.). Each group included 7–10 mice. These diets were continued for 22 weeks without any interruption. The food intake on the CDAA diet was monitored for 1 month, and similarity in food intake was observed between WT and TLR2−/− mice (Supporting Fig. 1).
To generate chimeric mice, liposomal clodronate was injected intravenously to deplete resident hepatic macrophages 1 day before bone marrow (BM) transplantation. BM cells (1 × 107 cells) obtained from WT or TLR2−/− mice were transplanted through tail veins after the recipient mice were lethally irradiated (10 Gy). The CDAA or CSAA diet was started 8 weeks after BM transplantation. Each group included 5–9 mice. The mice received humane care according to National Institutes of Health recommendations outlined in the Guide for the Care and Use of Laboratory Animals. All animal experiments were approved by the University of California San Diego and Akita University Institutional Animal Care and Use Committee.
Hematoxylin and eosin, Oil Red O, sirius red, and immunohistochemical staining for α smooth muscle actin (αSMA; Dako Cytomation, Kyoto, Japan), F4/80 (eBioScience, San Diego, CA), and Ly6C (Abcam, Cambridge, MA) were performed.9 Sirius red positive area was measured on 10 low-power (×40) fields/slide and quantified with the use of National Institutes of Health imaging software. NAFLD activity score was determined according to the published criteria.25 F4/80- and Ly6C-positive cells were counted on 10 high-power (200×) fields per slide.
RNA extracted from livers and cells was converted to complementary DNA via reverse transcription. Quantitative real-time polymerase chain reaction was then performed using an ABI PRISM 7000 Sequence Detector (Applied Biosystems, Foster City, CA). Genes were normalized to 18S RNA as an internal control. The primer sequences are summarized in Supporting Table 1.
Table 1. Body, Liver, and Fat Weight and Lipid Levels After 22 Weeks of CSAA and CDAA Diet Feeding
WT Mice (n = 7)
TLR2−/− Mice (n = 7)
WT Mice (n = 10)
TLR2−/− Mice (n = 7)
WT Mice (n = 10)
TLR2−/− Mice (n = 9)
All values are presented as mean ± SD.
Significantly different from WT mice on standard chow (P < 0.05).
Significantly different from WT mice on a CSAA diet (P < 0.05).
Significantly different from WT mice on a CDAA diet (P < 0.05).
Hepatic lipids were isolated as described.9 Triglyceride, total cholesterol, and FFA contents were measured using Triglyceride E (Wako, Osaka, Japan), Cholesterol E (Wako), and NEFA C-test (Wako) according to the manufacturer's instructions.
Protein extracts were electrophoresed and then blotted. Blots were incubated with antibody for αSMA (Sigma, St. Louis, MO) or caspase-1 (Millipore, Temecula, CA).
Measurement for Alanine Aminotransferase, Caspase-1 Activity, Insulin, IL-1α, IL-1β, and Tumor Necrosis Factor α.
A kit for measuring serum alanine aminotransferase (ALT) levels was obtained from Wako, and a caspase-1 activity assay kit (catalog #BF141000) was purchased from R&D (Minneapolis, MN). Enzyme-linked immunoabsorbent assay kits were used for measuring insulin (Shibayagi, Gunma, Japan), IL-1β, IL-1α, and tumor necrosis factor α (TNF-α) (eBioscience). Insulin resistance was assessed via the homeostasis model assessment of insulin resistance [immunoreactive insulin (μU/mL) × fasting blood sugar (mg/dL) ÷ 405].26
Cells and Treatment.
Kupffer cells, HSCs, and hepatocytes were isolated from mice as described.9 In experiments using hepatocytes, 200 μL of liposomal clodronate was injected intravenously 1 day before isolation to deplete contaminated Kupffer cells. We have confirmed the intact responses of these hepatocytes to TNF-α, IL-6, and insulin as assessed by activation of nuclear factor κB, signal transducer and activator of transcription 3, and Akt (data not shown). Hepatocytes and Kupffer cells were cultured in serum-free media, and HSCs were cultured with 1% fetal bovine serum containing Dulbecco's modified Eagle's medium for 16 hours before stimulation with reagents. Pam3CK4 (5 μg/mL) (EMC Microcollection, Tuebingen, Germany), CpG-ODN (5 μg/mL) (ODN1826: 5′-tccatgacgttcctgacgtt-3′) (Invitrogen), and palmitic acid (200 μM) (Sigma) were used to stimulate liver cells.
Differences between two groups were compared using a Mann-Whitney U test. Differences between multiple groups were compared using one-way analysis of variance (Dr. SPSS II); P < 0.05 was considered significant.
TLR2−/− Mice Exhibit Less Inflammation in CDAA Diet–Induced NASH.
As we have reported,9 mice on a CDAA diet for 22 weeks resulted in severe steatosis, inflammatory cell infiltration, and hepatocyte ballooning (Fig. 1A). TLR2−/− mice on a CDAA diet had similar grades of steatosis (Fig. 1A) and hepatic triglyceride content as WT mice (Table 1), whereas inflammatory cell infiltration and hepatocyte ballooning were markedly attenuated in TLR2−/− livers (Fig. 1A). Accordingly, the NAFLD activity score in TLR2−/− mice was significantly lower than that in WT mice (total score, WT versus TLR2−/− mice = 6.5 versus 4.6; P < 0.05) (Fig. 1A). WT livers on the CDAA diet increased infiltration of inflammatory cells that express F4/80 and Ly6C, while the recruitment of inflammatory cells was decreased in TLR2−/− livers (Fig. 1B). Serum ALT levels (Fig. 1C) and the expressions of proinflammatory cytokines and chemokines (Fig. 1D) were also decreased in TLR2−/− mice compared with WT mice. The WT mice fed the CDAA diet also displayed obesity and insulin resistance (Fig. 1E, F). While similar metabolic phenotypes were shown between WT and TLR2−/− mice fed on standard chow (Table 1), the changes in weight gain and insulin resistance were blunted in TLR2−/− mice on the CDAA diet (Fig. 1E, F and Table 1). Moderate steatosis was seen in control CSAA diet–fed mice, but inflammation was not evident in these animals (Fig. 1A-D).
Liver Fibrosis Is Suppressed in TLR2−/− Mice After CDAA Diet Feeding.
Liver fibrosis is a major manifestation of advanced NASH. The CDAA diet for 22 weeks resulted in perisinusoidal fibrosis in WT mice as assessed by sirius red staining (Fig. 2A, B). In contrast, TLR2−/− mice did not show significant liver fibrosis (Fig. 2A, B). αSMA expression was also attenuated in TLR2−/− mice as examined by immunohistochemistry (Fig. 2C) and immunoblotting (Fig. 2D). Hepatic messenger RNA (mRNA) levels of collagen α1(I), collagen α1(IV), transforming growth factor β1 (TGF-β1), tissue inhibitor of metalloproteinase-1 (TIMP-1), and plasminogen activator inhibitor-1 (PAI-1) were decreased in TLR2−/− mice compared with those in WT mice (Fig. 2E). Neither WT nor TLR2−/− mice fed a control CSAA diet developed liver fibrosis or HSC activation (Fig. 2 A -2E).
A TLR2 Ligand Activates Both Kupffer Cells and HSCs.
Because Kupffer cells and HSCs are the primary cells involved in hepatic inflammation and fibrosis, we examined whether cultured Kupffer cells and HSCs respond to a TLR2 ligand.27 Treatment with Pam3CK4, a synthetic TLR2 ligand, increased TNF-α levels in WT Kupffer cells at the mRNA (Fig. 3A) and protein levels (Fig. 3B). In both quiescent and culture-activated HSCs, Pam3CK4 treatment increased mRNA expression of fibrogenic genes, including TIMP-1, PAI-1, and TGF-β1 (Fig. 3C), and decreased mRNA expression of Bambi, a decoy receptor for TGF-β receptor signaling (Fig. 3D), demonstrating that TLR2 ligands induce a profibrogenic phenotype in HSCs.
Hematopoietic Cells, Including Kupffer Cells, Contribute to Progression of Liver Inflammation and Fibrosis.
Because both Kupffer cells and HSCs may contribute to TLR2-mediated NASH and fibrosis, we wanted to determine the responsible cells that express functional TLR2 for the development of steatohepatitis. We generated TLR2 BM chimeric mice, in which hepatic macrophages were reconstituted with transplanted BM cells. As reported, more than 90% of Kupffer cells were replaced with transplanted BM cells 3 months after BM transplantation.23,28–30 Mice transplanted with WT BM (WT BM cells transplanted to WT mice or TLR2−/− mice) on the CDAA diet showed severe steatosis and inflammatory cell infiltration (Fig. 4A). In contrast, mice transplanted with TLR2−/− BM (TLR2−/− BM cells transplanted to WT mice or TLR2−/− mice) had less inflammatory cell infiltration (Fig. 4A). Increases in serum ALT and hepatic mRNA levels of inflammatory cytokines were suppressed in mice transplanted with TLR2−/− BM cells (Fig. 4B, C). These results indicate that hematopoietic cells, including Kupffer cells, are the primary cell types involved in hepatic inflammation mediated by TLR2. Consistent with the histopathology of liver inflammation, liver fibrosis was attenuated in mice reconstituted with TLR2−/− BM compared with mice with WT BM as assessed by sirius red staining (Fig. 4A). Hepatic mRNA levels of fibrogenic genes were significantly suppressed in mice transplanted with TLR2−/− BM (Fig. 4D). Thus, hematopoietic cells, including Kupffer cells, but not HSCs, are responsible for TLR2-mediated liver inflammation and fibrosis in NASH.
TLR2 Ligand and Palmitic Acid Cooperatively Activate the Inflammasome in Kupffer Cells.
We sought to elucidate the mechanism by which Kupffer cells are the primary cell types in TLR2-mediated NASH. Because palmitic acid has been reported to be a TLR2 ligand,19,20 we tested whether palmitic acid can produce TLR2-mediated cytokines that are induced by Pam3CK4 (Fig. 3). In contrast to previous studies,18,19 WT Kupffer cells did not increase TNF-α and CD68 in response to palmitic acid, ranging from 10 to 500 μM (Supporting Fig. 2A, B). Furthermore, palmitic acid did not increase mRNA expression of fibrogenic factors in HSCs (Supporting Fig. 2C). These data indicate that palmitic acid alone would not function as a TLR ligand in Kupffer cells or HSCs.
Next, we tested whether palmitic acid contributes to inflammasome activation. Pam3CK4 increased mRNA expression of IL-1β and IL-1α in WT Kupffer cells, but palmitic acid did not (Fig. 5A, B). Neither Pam3CK4 nor palmitic acid increased IL-1β mRNA in HSCs (Supporting Fig. 3A). We then measured the active form of IL-1β and IL-1α in supernatant. In the presence of Pam3CK4, palmitic acid induced secretion of the active form of IL-1β and IL-1α from Kupffer cells (Fig. 5B, D). In contrast, HSCs did not secrete IL-1β by the same treatment (Supporting Fig. 3B), and palmitic acid or Pam3CK4 alone did not induce IL-1β secretion in either Kupffer cells or HSCs, suggesting that two-step stimulation with TLR2 ligand and palmitic acid is required for inflammasome activation. To assess inflammasome activation by TLR2 ligands and palmitic acid, we measured the expression of inflammasome components, including NLRP3, ASC, NLRP1, and AIM2. Among them, Pam3CK4 induced NLRP3 expression in Kupffer cells, but not in HSCs (Fig. 5C, Supporting Fig. 3C), and NLRP3 was not induced by palmitic acid treatment alone (Fig. 5C). In agreement with the aforementioned findings, Kupffer cells primed with Pam3CK4 increased caspase-1 activity in response to palmitic acid (Fig. 5D, E). As expected, palmitic acid did not activate caspase-1 in HSCs primed with Pam3CK4 (Supporting Fig. 3D). We further examined hepatocytes to respond to Pam3CK4 and palmitic acid. Primary hepatocytes isolated from mice pretreated with liposomal clodronate to deplete Kupffer cells in vivo did not increase levels of IL-1β mRNA and protein, NLRP3 mRNA, and caspase-1 activity by Pam3CK4 and palmitic acid treatment (Supporting Fig. 4A-D). TNF-α production was also not seen in these hepatocytes in response to Pam3CK4 (Supporting Fig. 4E, F). These results indicate that hepatocytes are not the cell type responsible for TLR2-mediated inflammasome activation.
Diminished Inflammasome Activation in TLR2−/− Mice.
Finally, we investigated inflammasome activation in vivo. WT mice on the CDAA diet increased expressions of IL-1β mRNA (Fig. 1F) and the inflammasome component NLRP3 in the liver (Fig. 6A). In contrast, this induction was diminished in TLR2−/− mice liver (Fig. 1F, 6A). Moreover, portal vein FFA levels were lower in TLR2−/− mice than in WT mice (Fig. 6B). In addition, WT mice on the CDAA diet exhibited increased hepatic caspase-1 activity and serum levels of IL-1β and IL-1α (Fig. 6C-E), whereas TLR2−/− mice had lower activity of caspase-1 in the liver and decreased IL-1β and IL-1α levels in sera (Fig. 6C-E). These results demonstrate that TLR2 is required for activation of the inflammasome in mice fed the CDAA diet.
The Collaborative Effect of TLR2 and TLR9 Ligands on Cytokine Production in Kupffer Cells.
Because TLR9 signaling promotes NASH progression through IL-1β,9 we investigated whether palmitic acids participate in TLR9-mediated IL-1 production. In combination with CpG-ODN, a synthetic TLR9 ligand, palmitic acids functioned to secrete IL-1α and IL-1β in Kupffer cells (Fig. 7A, B). Then, we examined the collaborative effect of TLR2 and TLR9 ligands in Kupffer cells. TLR2 and TLR9 ligands synergistically up-regulated mRNA levels of proinflammatory cytokines, including IL-1β, IL-1α, TNF-α, and monocyte chemoattractant protein 1 (MCP-1) (Fig. 7C-F). The synergistic effects of TLR2 and TLR9 ligands were also observed in the secretion of IL-1α and TNF-α proteins (Fig. 7A, B,E). These results suggest that TLR2 and TLR9 signaling collaboratively induce cytokine production in Kupffer cells that promote NASH.
We have previously demonstrated that activation of TLR4 signaling is crucial for the development of liver fibrosis through amplification of TGFβ signaling in HSCs.28 In murine NASH models, TLR4 ligand lipopolysaccharide (LPS) and TLR9 ligand bacterial DNA were elevated in the plasma,9,23 and the signaling of TLR4 and TLR9 promoted NASH development. MyD88, an adaptor molecule for TLR2, TLR4, and TLR9, is crucial for NASH progression.9 The present study demonstrates that TLR2 signaling acts as a promoter of liver inflammation and fibrosis in CDAA diet–induced NASH (Fig. 1 and 2). Interestingly, TLR2−/− mice displayed less liver inflammation and fibrosis but similar levels of hepatic steatosis as WT mice, indicating that TLR2 signaling contributes to liver inflammation and fibrosis, but that hepatic lipid accumulation is independent of TLR2. Although a TLR2 ligand can activate both Kupffer cells and HSCs (Fig. 3), our in vivo study using TLR2 BM chimeric mice demonstrated that Kupffer cells are responsible for TLR2-mediated liver inflammation and fibrosis (Fig. 4). Activation of the inflammasome and the production of IL-1α and IL-1β in WT mice on the CDAA diet were suppressed in TLR2-deficient mice (Fig. 6). In Kupffer cells, the cooperative actions mediated by TLR2 ligand and palmitic acids are required for activation of the inflammasome and production of IL-1α and IL-1β (Fig. 5). We also found that TLR2 and TLR9 signaling synergistically induce production of inflammatory cytokines that can promote NASH (Fig. 7).
A previous study reported that TLR2 deficiency aggravates NASH developed on an MCD diet.12 When mice are fed an MCD diet, TLR2−/− mice are more susceptible to LPS-induced inflammatory and fibrogenic responses than WT mice.7,12 The present study used the CDAA diet that produces weight gain, insulin resistance, and liver fibrosis, in contrast to MCD diet that causes weight loss, improvement of insulin signaling, and only very mild liver fibrosis. Our data showed that the portal vein FFA levels were elevated in mice fed the CDAA diet, but not the MCD diet (Supporting Fig. 5). Because the majority of NASH patients develop obesity, insulin resistance, and liver fibrosis with elevation of FFA levels,21 we propose that the CDAA diet model is more relevant to human NASH than the MCD diet model. The murine HFD model induces obesity, insulin resistance, and hepatic steatosis, but liver inflammation is very mild and fibrosis does not develop. TLR2−/− mice and MyD88−/− mice had less steatosis than WT mice on a HFD.10,11 Similarly, TLR2−/− mice exhibited less hepatic inflammation and fibrosis on the CDAA diet, which influences obesity and systemic insulin resistance. Notably, TLR2−/− mice on the CDAA diet developed a similar degree of hepatic steatosis with WT mice, but insulin resistance was suppressed. Alternatively, the CSAA diet caused much less hepatic steatosis than the CDAA diet but induced a similar degree of insulin resistance caused by the CDAA diet (Fig. 1).9,31 These findings suggest that systemic insulin resistance, simple steatosis, and NASH substantially affect one another, but could be caused by different etiologies, and steatosis is not a preliminary hepatic condition for NASH.32
The inflammasome is a multiprotein platform containing NLRP1, NLRP3, NLRC4, and AIM2 that activates caspase-1 through the adaptor protein ASC. Activated caspase-1 proteolytically cleaves pro–IL-1β and pro–IL-18 to generate active forms of IL-1β and IL-18.33 A recent report demonstrated that IL-1α secretion also requires inflammasome activation.34 Inflammasome activation has been reported to be important for the development of obesity, hepatic steatosis, and insulin resistance.13,14,16 In these reports, NLRP3−/−, ASC−/−, and caspase-1−/− mice on an HFD had less hepatic steatosis and obesity and improved insulin sensitivity.13,14,16 The function of the inflammasome in a NASH model induced by an MCD diet is controversial. Two reports have shown that activation of inflammasome components, including caspase-1, promotes liver inflammation and fibrogenesis in NASH induced by an MCD diet.17,35 However, a recent study demonstrated that mice deficient in caspase-1, ASC, or NLRP3 developed more steatosis while on an MCD diet.36 The results from our present and previous studies are in agreement with the concept that inflammasome activation promotes NASH and insulin resistance. In particular, TLR2−/− mice fed a CDAA diet exhibited decreased expression of inflammasome components, including NLRP3, caspase-1, IL-1α, and IL-1β, and TLR2−/− mice and IL-1R−/− mice showed reduced body weight gain and suppression of insulin resistance, liver injury, inflammation, and fibrosis compared with WT mice.9 TLR ligands alone are sufficient for induction of NLRP3 through nuclear factor κB, but are insufficient to activate the inflammasome.37,38 Additional factors such as particular bacterial toxins, extracellular adenosine triphosphate, or certain danger signals are required for assembly of inflammasome components and activation.33,39,40 Dietary factors, such as uric acids, cholesterol crystal and palmitic acid were also reported as a trigger to assemble the inflammasome components in macrophages.16,41,42 Wen et al. demonstrated that LPS activates the inflammasome in cooperation with palmitic acid, one of the most abundant saturated fatty acids in the plasma of NAFLD, in BM-derived macrophages.16,42 Similarly, our data demonstrated that the TLR2 or TLR9 ligand alone failed to activate the inflammasome, while palmitic acid in concert with a TLR2 or TLR9 ligand induced inflammasome activation in Kupffer cells (Figs. 5 and 7).9 Importantly, in hepatic macrophages/Kupffer cells, LPS alone can activate the inflammasome to produce IL-1β, IL-1α, and IL-18 through the MyD88-independent TRIF-dependent pathway (Fig. 7A, B).43,44 Although palmitic acid has been characterized as a TLR2 and TLR4 ligand to induce inflammatory cytokines,18–20 data from recent research and ours did not support this effect of palmitic acid in both Kupffer cells and HSCs.45
HMGB-1 released from damaged hepatocytes may activate TLR2 in addition to TLR4 as an endogenous ligand in NASH. Intestinal microflora can also be the source of TLR2 ligand because of contradictory phenotypes in metabolic disease in TLR2−/− mice between SPF and non-SPF conditions that affect the composition of gut flora.10,11,46 Oral bacteria contribute to the progression of metabolic disease, including atherosclerosis.47,48 Thus, endogenous ligands, such as HMGB-1, and the components derived from intestinal microflora or oral bacteria can be a ligand for TLR2 in NASH.
The present study demonstrates that TLR2−/− mice exhibited reduced NASH compared with WT mice, but seemed to have greater NASH pathology compared with TLR9−/− mice.9 Our data suggest that TLR2−/− Kupffer cells are more sensitive to produce inflammatory cytokines than WT Kupffer cells in response to TLR9 ligand, whereas TLR9−/− Kupffer cells have a response similar to TLR2 ligand compared with WT Kupffer cells (Fig. 7). It is conceivable that TLR9 signaling may compensates the expression of inflammatory cytokines in TLR2−/− Kupffer cells, which may explain the mechanisms underlying suppressed NASH pathology in TLR9−/− mice compared with TLR2−/− mice. However, liver inflammation in TLR2−/− mice was still less than WT mice, because NASH progression requires the synergy of TLR2 and TLR9 signaling. This synergy induces the production of inflammatory cytokines in Kupffer cells that promote NASH. In particular, TLR2 and TLR9-mediated IL-1 induces fibrogenic responses, including the up-regulation of TIMP-1 and PAI-1 and the down-regulation of Bambi, an endogenouse inhibitor of TGF-β; thus, the combination of these responses promotes liver fibrosis in HSCs.9
We thank Rie Seki, Karin Diggle, Jingyi Isabelle Song (Department of Medicine, University of California, San Diego), and Yukie Komatsu (Akita University Graduate School of Medicine) for excellent technical assistance.