Pathogen- or damage-associated molecular patterns during nonalcoholic fatty liver disease development

Abstract

Sensing danger is a key to an organism's survival. The appropriate reaction is immediate and effective and consists of triggering molecular and cellular events that lead to elimination of the menace with tolerable collateral damage, through the process of inflammation. Invasive microbes cause an immediate inflammation with recruitment of innate immune cells to the site of pathogen entry, activation of phagocytosis, release of chemokines and cytokines, and initiation of the adaptive immune response. The same mechanisms are activated when tissue damage occurs because of trauma, ischemia/reperfusion injury, chemical toxicity, and cellular necrosis, all events that should be followed by wound-healing to ensure the maintenance of body and organ integrity. Also, in this case, cells of the innate immune system rush to the damaged area, releasing proinflammatory cytokines and chemokines to cause a phenomenon called sterile inflammation. If the trigger cannot be removed and the process becomes chronic, both infective and sterile inflammation cause substantial collateral damage. The continuous but ineffective actions of the innate immune system perpetuate tissue damage, with collagen deposition, fibrosis, and eventually permanent anatomical and functional alterations.1 The organism senses microbial infection through innate receptors encoded in the genome, called pattern-recognition receptors, including the Toll-like receptors (TLRs), the nucleotide-binding and oligomerization domain (NOD)-like receptors, and retinoic acid–inducible gene I (RIG-I)-like receptors. These receptors recognize pathogen-associated molecular patterns (PAMPs) expressed by bacteria, fungi, and viruses, but also bind damage-associated molecular patterns (DAMPs), which are molecules released by sterile injury. Thus, PAMPs and DAMPs that bind to the same type of receptors initiate identical intracellular pathways terminating in identical effector functions. Each member of the PAMP and DAMP families binds to one or more different TLRs (Table 1), and TLR expression varies among disparate cell types. Thus, the same PAMP or DAMP may have different effector functions depending on the cell type with which the interaction takes place. TLRs are type 1 transmembrane glycoproteins consisting of leucine-rich repeat motifs in the extracellular domain for ligand recognition, and a cytoplasmic Toll/interleukin-1 (IL-1) receptor (TIR) intracellular domain indispensable for the activation of downstream signaling molecules. All TLRs ultimately activate the transcription factors nuclear factor kappa B (NF-κB) and activator protein-1 (AP-1) and thus the transcription of type 1 interferons and inflammatory cytokines. The proximal events of TLR signaling are initiated by the interaction of the TIR domain with cytosolic adaptor molecules. Myeloid differentiation factor 88 (MyD88) is the adaptor used by most TLRs, indispensable for the recruitment of the IL-1–associated kinases (IRAK1 and IRAK4). MyD88 is sufficient for signal transduction from TLR5, TLR7, TLR8, TLR9, and TLR11, but needs the cooperation of another adaptor, TIRAP (TIR domain–containing adaptor protein), downstream of TLR1-2, TLR2-6, and TLR4. Among all TLRs, TLR4 is the most peculiar, because it needs an accessory molecule, the glycoprotein MD-2, in order to effectively bind its best-known ligand, the bacterial endotoxin (lipopolysaccharide [LPS]). In addition, two independent signaling pathways are initiated by TLR4. Besides the TIRAP/MyD88-initiated cascade, a parallel signal runs through the TRAM (TRIF-related adaptor molecule) and Trif (TIR-domain–containing adapter-inducing interferon-β) adaptors, leading to the, albeit delayed, production of type 1 interferons.2 Stimulation of TLR4 via PAMPs such as LPS has been recently shown to play a central role in the development and progression of nonalcoholic fatty liver disease (NAFLD).3 On the contrary, the potential effect of DAMPs in NAFLD is still largely unexplored. In this issue of HEPATOLOGY, the study by Li et al. provides new, relevant data on the role of nuclear factor high mobility group box 1 (HMGB1) protein in mediating the activation of TLR4 signaling in hepatocytes in the early stage of NAFLD in mice.4 In particular, in their study, the authors investigate the role of the hepatocytic TLR4 during early NAFLD. Mouse transgenic models (mouse knockouts for TLR4, TLR2, MyD88, and TRIF) and wild-type mice were fed a normal or high-fat diet (HFD), and NAFLD parameters (i.e., steatosis, alanine aminotransferase levels, and the like), as well as potential proinflammatory activity of transcription factors such as p65-NF-κB, AP-1, and LITAF (LPS-induced tumor necrosis factor-alpha [TNF-alpha] factor) were investigated. Contrary to previous studies,3 the authors report that Kupffer cell depletion only partially prevents HFD-induced hepatocyte damage in wild-type mice, suggesting an important but not imperative role for these cells in the early stages of NAFLD. From their results, the authors conclude that TLR4/MyD88 signaling in liver parenchymal cells plays a pivotal role during the early phase of HFD-induced NAFLD in mice. DAMP, damage-associated molecular pattern; FFA, free fatty acid; HFD, high-fat diet; HMGB1, high mobility group box 1; HSCs, hepatic stellate cells; IL, interleukin; LPS, lipopolysaccharide; MyD88, myeloid differentiation factor 88; NAFLD, nonalcoholic fatty liver disease; NF-κB, nuclear factor κB; PAMP, pathogen-associated molecular pattern; TLR, Toll-like receptor. In addition, Li et al. demonstrate that increased hepatocytic expression and release of HMGB1 serves as trigger of TLR4/MyD88 signaling and inflammation in response to free fatty acid (FFA) infusion or HFD. HMGB1 was originally discovered as a nuclear protein that binds to nucleosomes to stabilize DNA structure and modulate transcriptional activity5; yet, when it is released or secreted after injury, HMGB1 meets all of the criteria for DAMPs. An important question is whether HMGB1 can promote, directly via TLR4 or indirectly by forming specific complexes with other molecules, the secretion of inflammatory molecules (cytokines and chemokines) in cells different from peripheral blood mononuclear cells. Interestingly, Li et al. report that neutralization of HMGB1 protects hepatocytes against FFA-induced TNF-alpha and IL-6 production, thus implicating hepatocytes as the direct target. On the basis of their results, Li et al. propose a model to explain the possible role of HMGB1 in FFA/HFD-induced NAFLD. In this model, exposure to FFA/HFD enhances HMGB1 intracellular expression and/or extracellular release via unknown mechanisms, activating TLR4/MyD88 signaling in hepatocytes, which in turn becomes self-sustaining in both parenchymal and nonparenchymal cells. The multifaceted pathology of NAFLD ranges from steatosis to steatohepatitis, which may progress to fibrosis and cirrhosis. Steatosis, necroinflammation, and ballooning are the core features of nonalcoholic steatohepatitis, but fibrosis represents the main tissue damage in NAFLD.6 Recently, there is accumulating evidence that TLR4-induced activation and sensitization of hepatic stellate cells (HSCs), because they regulate extracellular matrix destruction/reconstitution and tissue remodeling, may constitute the molecular link between hepatic inflammation and fibrogenesis in several chronic liver diseases, including NAFLD.7 Therefore, as demonstrated by in vitro experiments, it is conceivable that HSCs may also be targeted and addressed by HMGB1 toward a profibrogenic phenotype.8 This is in agreement with a recent study demonstrating that HMGB1 might be used as a noninvasive and informative marker of fibrosis grade, because its serum levels were significantly higher in patients who had chronic hepatitis B with low fibrosis (fibrosis score 1-2) compared to those with high fibrosis (fibrosis score 3-4).9 These findings highlight possible autocrine/paracrine effects of HMGB1 on different liver-resident cells. Indeed, although the study by Li et al. has significantly improved our knowledge of the possible role of HMGB1 in NAFLD, many issues remain unsolved. Is HMGB1 the sole DAMP that participates in inflammation and liver damage in NAFLD? Certainly, other DAMPs might be added to the list, including low-molecular-weight hyaluronic acid and defensins, because they (as well as other unidentified molecules) correlate with the presence of liver damage in NAFLD.10, 11 Furthermore, due to the multifactorial nature of NAFLD pathogenesis, upstream mechanisms and signals that lead to release of circulating PAMPs and DAMPs remain poorly delineated. In summary, the network of interactions that links PAMPs and DAMPs to TLR4 localized at the plasma membrane of several liver-resident cells and possible downstream signals might be more complicated than those proposed by Li et al. We have condensed our model of this network in Fig. 1. PAMP/DAMP and TLR4 signaling in liver-resident cells during NAFLD development. Genetic background, diet, and sedentary lifestyle strongly influence the circulating pattern of PAMPs and DAMPs by interfering with the gut–adipose tissue–liver axis. In liver-resident cells (hepatocytes, Kupffer cells, hepatic stellate cells [HSCs], and lymphocytes), the PAMP/DAMP engagement of TLR4 induces the activation both Trif- and MyD88-dependent pathways, of which the former requires TRAM and the latter requires TIRAP. Through mitogen-activated protein kinases, MyD88 and Trif promote phosphorylation and nuclear translocation of different transcription factors (TFs), such as AP-1, NF-κB, LITAF, and IRF3, which bind positive regulatory domains of genes encoding for several inflammatory molecules (IMs). These, in turn, may directly or indirectly create a vicious cycle that sustains the activation of TRL4 and/or other receptors (ORs) potentially involved in progression of liver damage during NAFLD. IMs maintain and increase tissue damage, and also recruit cells of the innate and adaptive immune system to the site of sterile inflammation. The possibility of targeting these molecules at different levels (i.e., diet supplementation interfering with the intestinal microbiota, pharmacological TLR inhibitors, and so forth) will open up new therapeutic opportunities for the treatment of NAFLD. However, toward this end, the role of PAMPs and, in particular, DAMPs in NAFLD needs to be investigated more thoroughly. It should be kept in mind that although blocking these molecules may represent a potential treatment to prevent and/or reverse liver damage, it represents a significant risk for the organism because these molecules are important elements of defense against infections.12