Innate immunity and hepatic inflammation

Innate immunity and hepatic inflammation With an average weight of 1.5 Kg, the liver represents the biggest internal organ of healthy adults (the skin being the largest of all with an average mass of 10 Kg) and receives blood supplies mainly via the portal vein and the hepatic artery (accounting for ~80% and ~20% of incoming circulation, respectively) [1]. Via these routes, the hepatic parenchyma not only is provided with oxygen and nutrients for its own needs, but also receives endogenous metabolic products, alimentary components, xenobiotics and microorganismal poisons for detoxification. Furthermore, the portal vein constitutes the major route through which metastatic cells attain the liver, de facto representing one of the most common sites for the metastatic spread of gastrointestinal, pancreatic, breast and ovarian neoplasms [2]. The liver is therefore constantly exposed to antigens and toxins that are capable of stimulating innate immune responses, whose function is to protect hepatic and systemic homeostasis against metastatic colonization or microbial pathogens and to contribute to the metabolic functions of liver by stimulating the detoxification of toxins and dietary products [3]. In normal conditions, innate immune responses in the liver are mainly mediated by the combined action of tissue-resident cells, including macrophages, Kupffer cells, dendritic cells, natural killer (NK) cells and natural killer T (NKT) cells, and circulating molecules, encompassing cytokines, chemokines, acute phase proteins and complement factors. However, circulating leukocytes such as monocytes can also be recruited to the hepatic parenchyma. Once in the liver, monocytes - most of which originate in the bone marrow from common myeloid progenitors [4] - generate tissueresident macrophages or myeloid dendritic cells, sharing with each other various surface markers including CD14 and CD16 [5]. The pristine role of these cells, which are activated by "microbeassociated molecular patterns" (MAMPs), i.e., basic components of bacteria and viruses that are not found in eukaryotic cells such as lipopolysaccharide (LPS) and double-stranded RNA, is to mount a relatively non-specific antimicrobial response involving phagocytosis as well as the secretion of pro-inflammatory cytokines, reactive oxygen species (ROS) and reactive nitrogen species (RNS) [6,

7]. In addition, macrophages and dendritic cells can respond to "damage-associated molecular patterns" (DAMPs), i.e., intracellular components of eukaryotic cells that are released/exposed only in situations of danger such as mitochondrial DNA or N-formyl peptides, thus mounting a “sterile” inflammatory response (see Main Article) [8, 9]. Sterile inflammation contributes to the maintenance of tissue homeostasis both as it participates in the initiation of cognate immune responses against malignant cells [10], and as it promotes the removal of senescent cells, apoptotic corpses and cellular debris [11]. Of note, MAMPs and DAMPs are detected by the innate immune system via a common set of pattern recognition receptors (PRRs), including transmembrane Tolllike receptors (TLR) as well as intracellular sensors such as NOD-like receptors [12]. Macrophages exist in at least two, functionally and phenotypically distinct states [11]. On one hand, “classically” activated (M1) macrophages develop in the presence of interferon γ (IFNγ) and LPS, express high levels of HLA-DR, and predominantly stimulate interleukin (IL)-12-associated helper T-cell responses (i.e., T H 1 responses) by secreting IL-1, IL-6 and tumor necrosis factor α (TNFα), thus exerting robust microbicidal and antineoplastic effects. On the other hand, “alternatively” activated (M2) macrophages originate in response to IL-4 and IL-13, bear on their surface consistent amounts of CD163 and CD206 and mainly promote T H 2 responses (as they secrete elevated quantities of IL-10), hence stimulating tissue remodeling and angiogenesis [11, 13]. Kupffer cells, which de facto constitute liver-resident macrophages, also secrete a plethora of proinflammatory mediators including IL-1, IL-6, TNFα, granulocyte macrophage colony-stimulating factor (GM-CSF), several chemokines, ROS and nitric oxide (NO). An excessive or chronic secretion of pro-inflammatory molecules by M1 macrophages and Kupffer cells can induce the apoptotic or necrotic demise of hepatocytes. In these conditions, liver injury is often associated to a fibrotic response that results from the excessive deposition of extracellular matrix components by hepatic stellate cell (HSC)-derived myofibroblasts and portal fibroblasts [14, 15]. Importantly, hepatic fibrosis can be limited by macrophage depletion during the injury phase, but not during the

repair phase, of CCl 4 -driven inflammation, indicating that functionally distinct subpopulations of macrophages play opposing roles throughout this process [16]. Similar to monocytes, eosinophils differentiate in the bone marrow from common myeloid progenitors, mostly in response to IL-3, IL-5 and GM-CSF [17]. Circulating eosinophils can be recruited to the hepatic parenchyma in response to various conditions, including (but not limited to) parasitic and viral infections, allergic reactions and hypersensitivity to drugs [18]. Intra-hepatic eosinophils can be activated by IL-5 and several TLR agonists to release into the extracellular space the content of their granules, i.e., highly reactive cationic proteins such as major basic protein (MBP), eosinophil-derived neurotoxin (EDN), eosinophil cationic protein (ECP) and eosinophil peroxydase (EP) [17]. These factors not only exert potent microbicidal functions but also can kill virus-infected and malignant cells [17]. Cell death induction by eosinophils is thought to contribute to the maintenance of hepatic homeostasis as it promotes immunological tolerance. However, the persistent presence of activated eosinophils mediates hepatotoxic effects and these cells have also been suggested to execute a non-canonical pathway of liver allograft rejection [19]. Similar observations hold true for various classes of NK and NKT cells, the latter of which are particularly abundant in the liver. Thus, NK and NKT recruited to the hepatic parenchyma during hepatitis B virus (HBV) infection contribute to inflammation by releasing cytokines such as IL-4 and IFNγ [20] and by inducing the death of both virus-infected and bystander hepatocytes in a TNFrelated apoptosis-inducing ligand (TRAIL)-dependent fashion [21]. However, such an initially beneficial activity of NK and NKT cells can become harmful for the hepatic parenchyma and drive fibrosis if not properly controlled by the combined action of various cytokines and chemokines [22]. Thus, mild inflammatory responses (that is, limited in intensity and duration) appear to be beneficial for the liver as they eliminate noxious stimuli, favor tissue repair and contribute to the reestablishment of homeostasis. Conversely, excessive or chronic inflammation is often associated

with massive hepatocyte death and with the initiation of fibrosis, as a result of a complex interplay between liver-resident cells, cells that infiltrate the hepatic parenchyma in response to stress and circulating factors. During the last decade, an intense experimental effort has been dedicated at understanding this crosstalk, revealing the essential causes and mechanisms of hepatocyte death during inflammation.

Cell death modalities in the liver According to current guidelines, apoptosis and necrosis represent the two extremes of a spectrum of cell death morphotypes [23]. First described in the 1960s by Richard Lockshin and John Kerr, apoptosis generally manifests with a reduction of cellular volume (pyknosis) coupled to chromatin condensation, nuclear fragmentation (karyorrhexis), little ultrastructural modifications of organelles and a characteristic “blebbing” of the plasma membrane [24]. Although necrosis was known to pathologists well before apoptosis, as it was often observed in the context of accidental injuries and chronic pathologies associated with massive tissue damage (e.g., alcoholic cirrhosis), it is only during the last decade that the stereotyped morphological nature of necrosis has begun to emerge [23]. Thus, necrotic cells often exhibit an increasingly translucent cytoplasm, prominent organellar swelling, minor ultrastructural modifications of the nucleus and an increased cell volume (oncosis), culminating in the breakdown of plasma membranes. Moreover, at odds with apoptosis, necrosis is not accompanied by the fragmentation of dying cells into discrete corpses [25]. The apoptosis-necrosis dichotomy has permeated the cell death research field until very recently. Thus, while apoptosis has long been viewed as a physiological, immunologically silent (if not tolerogenic) and genetically regulated cell death mode, also owing to the pioneering work of Robert Horvitz in Caenorhabditis elegans [26], necrosis was tagged as pathological, pro-inflammatory and accidental [24]. The relatively recent discovery of immunogenic instances of apoptosis [10] and the characterization of a complex signal transduction cascade that eventually leads to necrosis [10] have now made clear that considering apoptosis and necrosis as diametrically opposed entities at a functional level constituted a gross oversimplification. Schematically, apoptosis can ensue the activation of two distinct signal transduction cascades (Figure 1). On one hand, the “extrinsic” pathway transduces extracellular signals of stress into a cell death response either via death receptors (e.g., FAS, TNF receptor 1 [TNFR1]), which respond to specific molecules (e.g., FASL, TNFα), or via “dependence receptors” (e.g., PTCH1, deleted in

colorectal carcinoma [DCC]), which are activated when the concentration of their ligands falls below a critical threshold level [27]. Ligand bound death receptors normally transduce apoptotic signals as they promote the proximity-induced activation of caspase-8 (or -10), hence setting off apoptosis execution by caspase-3, -6 and -7. This is not the case of dependence receptors, which appear to mediate pro-apoptotic effects either by favoring the activation of caspase-9 or via deathassociated protein kinase 1 (DAPK1) [27]. On the other hand, the “intrinsic” or “mitochondrial” pathway is activated by a variety of intracellular stress conditions that cells are unable to cope with (e.g., DNA damage, ROS overproduction, Ca2+ overload, viral infection, …) [28]. Mitochondria play a critical role in this setting, as they constitute the battlefield where pro- and anti-apoptotic signals are opposed to each other, de facto regulating the rate-limiting step of intrinsic apoptosis [28]. The widespread permeabilization of mitochondrial membranes constitutes indeed a point-ofno-return in this process, as it results in (i) the release of direct (e.g., cytochrome c) and indirect (e.g., direct IAP-binding protein with low pI [DIABLO]) activators of caspases as well as of caspase-independent cell death effectors (e.g., apoptosis-inducing factor) into the cytosol [29], and (ii) the dissipation of the mitochondrial transmembrane potential (Δψ m ), in turn provoking the cessation of mitochondrial ATP synthesis as well as all other Δψ m -dependent metabolic functions [30]. Of note, while most of the phenotypic traits of apoptosis stem from the proteolytic activity of caspases, at least in some settings, mitochondrial outer membrane permeabilization (MOMP)dependent instances of cell death proceed normally even in the presence of caspase-blockers [27]. MOMP can originate either at the mitochondrial outer membrane, owing to the pore forming capacity of pro-apoptotic members of the BCL-2 protein family such as BAX and BAK [31], or at the inner mitochondrial membrane, due to the activity of a supramolecular entity known as “permeability transition pore complex” (PTPC) [28]. A detailed description of the molecular cascades leading to MOMP largely exceeds the scope of this review and can be found in Refs [28, 32]. This said, it should be noted that the molecular machineries for extrinsic and intrinsic apoptosis are not fully disjointed, as (i) in some cell types (including hepatocytes), the caspase-8-mediated

cleavage of the BH3-only protein BID generates a fragment that promotes MOMP [33], and (ii) the ligation of death receptors normally promotes an oxidative burst that may stimulate the opening of the PTPC [28]. For a long time, intracellular ATP levels have been viewed as the main molecular switch between (genetically controlled) apoptosis and (unregulated) necrosis. At odds with the latter, the former was indeed thought to require a minimum of energy supplies [34]. This concept has definitely been abandoned along with the discovery of regulated instances of necrosis, including necroptosis [25, 35]. The term necroptosis has been introduced in 2005 by Junying Yuan and colleagues to indicate a non-apoptotic form of cell death that can (i) ensue the ligation of TNFR1 in caspase-deficient conditions, and (ii) can be inhibited by the small molecule necrostatin-1 [36]. A few years later, the receptor-interacting protein kinase 1 (RIPK1) was found to constitute the molecular target of necrostatin 1, and since then several other components of the signal transduction cascade leading to necroptosis have been identified, including (but not limited to) the RIPK1 homologue RIPK3, the cytosolic kinase mixed lineage kinase domain-like (MLKL) and the mitochondrial phosphatase phosphoglycerate mutase family member 5 (PGAM5) [25, 37].

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