Necroptosis is a programmed cell death first discovered in 2005 (5). It can be activated by members of the tumor necrosis factor (TNF) family (through TNF receptor (TNFR) 1, TNFR2, TRAILR1, and TRAILR2), Fas ligand, toll-like receptors (TLR), lipopolysaccharides (LPS), double-stranded RNA (dsRNA), and genotoxic stress. Furthermore, different physical-chemical stress stimuli can also initiate necroptosis, including ATP-depletion, radiation, ischemia-reperfusion injury, glutamate, and lipid overload (5, 6).

The necroptosis signaling pathway is primarily modulated by activation of receptor-interacting protein kinase (RIPK) 3, which phosphorylates mixed-lineage kinase domain-like protein (MLKL), mediating MLKL oligomerization. In the absence of caspase-8, RIPK1 recruits and phosphorylates RIPK3 to form the ripoptosome and then phosphorylates MLKL to form the necrosome (a complex containing RIPK1, RIPK3, and MLKL). After RIPK3 phosphorylates MLKL, p-MLKL undergoes conformational changes and oligomerization and is transferred to phosphatidylinositol-rich patches on the plasma membrane to form macropores. This can lead to necroptotic cell death by allowing ion influx, cell swelling, and membrane rupture, followed by the uncontrolled release of intracellular material, mainly DAMPs (7, 8). In addition to RIPK1-independent activation, necroptosis can be also triggered by activation of TLR3 and TLR4 by dsRNA and LPS respectively, through TIR-domain-containing adaptor-inducing interferon-β (TRIF)-dependent activation of RIPK3. Viral RNA and the DNA/RNA released from damaged mitochondria (mtDNA/mtRNA) can induce necroptosis by Z-DNA binding protein 1 (ZBP1)-dependent activation of RIPK3. Activated RIPK3 then phosphorylates MLKL (9). From this, it can be concluded that RIPK3 and MLKL are integral components of necroptosis regardless of their upstream trigger ( Figure 1 ).

DAMPs secreted from necroptotic cells have been identified as potent activators of the immune system that trigger fibrosis. Necroptotic DAMPs can be divided into two categories (1): molecules that perform noninflammatory functions in living cells (such as HMGB1) and acquire immunomodulatory properties when released, secreted, modified, or exposed on the cell surface during cellular stress, damage, or injury, or (2) alarmins, molecules that possess cytokine-like functions (such as IL-1α, S100A8, and IL-33) that can be stored in cells and released upon cell lysis, whereupon they contribute to the inflammatory response (10).

Necroptotic DAMPs can directly activate profibrotic responses of nonimmune cells, such as epithelial cells, endothelial cells, and fibroblasts. For example, necroptotic macrophages are reported to promote collagen synthesis in fibroblasts via the release of IL-6 and TNF-α during fat graft fibrosis (11). Experiments have also shown that self-DNA produced by necroptotic glomeruli and tubules in the kidney activates cyclic guanosine monophosphate-adenosine monophosphate synthase and is absent in melanoma 2, thereby inducing fibroblast proliferation and migration (12, 13).

In addition, the innate immune response is proposed to contribute to the pathogenesis of fibrosis (2). Necroptotic DAMPs, including ATP and HMGB1, can activate innate immune cells such as neutrophils and macrophages. Activation of these cells leads to the production of various cytokines and chemokines, which in turn recruit inflammatory cells and promote collagen secretion in fibroblast or hepatic stellate cells (HSCs) (14). In addition, group 2 innate lymphoid cells can produce more IL-13 when exposed to IL-33, which leads to the activation of fibroblasts or HSCs.

The recognition of DAMPs relies on the cell surface, endosomal, and cytosolic pattern recognition receptors (PRRs) that include TLRs, NOD-like receptors (NLRs), C-type lectin receptors (CLRs), and receptors for advanced glycation end products (RAGE). These DAMP-sensing receptors, together with inflammasomes, might represent an important pathway responsible for converting self-limited regenerative repair into an unresolved fibrotic process (15) ( Figure 2 ).

Necroptotic DAMP receptors have also been reported to promote fibrosis. For example, in ischemia-reperfusion injury animal models, gene knockout or inhibition of TLR can alleviate systemic inflammation and tissue fibrosis in the liver, kidney, and intestine (16). Macrophage-inducible C-type lectin is a CLR that mediates collagen secretion in macrophages during adipose tissue fibrosis (17). Moreover, NLRs and other PPPs exhibit significant interplay. Some NLRs can detect DAMPs and stimulate the expression of proinflammatory genes, while other NLRs require the participation of other PRRs in order to promote the processing and secretion of the key proinflammatory cytokines, IL-1β and IL-18, which are involved in the pathogenesis of various inflammatory diseases (including systemic sclerosis (SSc), atherosclerosis, and arthritis), leading to tissue fibrosis (18).

Inflammasomes are the bridge signaling between DAMP identification and the immune response. According to the differently activated caspases, inflammasomes are divided into two categories: classical and non-classical. Classical inflammasomes mainly activate caspase-1, including NOD-like receptor protein 1 (NLRP1), NOD-like receptor protein 3 (NLRP3), NOD-like receptor C4 (NLRC4), and NLRC5; nonclassical inflammasomes mainly activate caspases other than caspase-1, including AIM2, etc. (19). The NLRP3 inflammasome, the most studied, is typically composed of NLRP3, ASC, and procaspase-1. The inflammasome complexes induce cells to produce cytokines. Cumulative evidence shows that inflammasomes are involved in the formation of fibrosis in multiple organs (20–22). Thus, the next specific introductions will be made according to different receptors and their corresponding inflammasomes.

Next, specific introductions will be made according to different receptors and their corresponding inflammasomes:

The wound-healing process is divided into three stages: inflammation, regeneration, and remodeling. Keratinocytes and dermal fibroblasts are thought to be key cells in wound re-epithelialization and closure. Recently, necroptotic cell death has been observed at the site of chronic wounds (92). Khandelwal et al. reported that protecting keratinocytes from necroptosis using a surfactant polymer wound dressing can favor wound healing (25). Another study showed that RIPK3 deficiency fully prevents skin lesion development in animals with epidermal keratinocyte-restricted deficiency of the adaptor protein FAS‐associated death domain by inhibiting deubiquitinase enzyme cylindromatosis and TNFR1 signaling (92). In addition, FAS-associated death domain or caspase-8 deficiency causes RIPK3-dependent keratinocyte necroptosis and induces the release of IL-1α, which worsens skin inflammation (93). HMGB1, an ubiquitous nuclear protein, is present in almost all cell types in the nucleus and cytoplasm. The release of HMGB1 from necroptotic keratinocytes has been observed during chronic wound healing (25). The inflammatory function of HMGB1 is mediated by its binding to cell-specific receptors such as RAGE, TLR2, TLR4, and TLR9. Serum HMGB1 levels are higher in patients with scleroderma and are correlated with skin fibrosis (94). Reactive oxygen species–induced dermal fibroblast necroptosis has also been observed in skin wound healing in diabetes. Sirtuin 3 activation has been suggested as a potentially promising therapeutic strategy for skin wound healing in diabetes through the prevention of fibroblast necroptosis (95). According to these findings, necroptosis exerts a damaging effect on wound healing. In addition, chronic wound healing may be accelerated by the expression of essential regulatory elements in necroptosis.

Liver fibrosis is closely associated with chronic liver disease. Cell death in chronic liver disease has been reported to contribute to chronic hepatocyte turnover, recruitment of immune cells, activation of hepatic stellate cells, and, thereby, the development of liver fibrosis (96).

In steatohepatitis (NASH), RIPK3-mediated necroptosis has been shown to be upregulated in both human and dietary-related mouse NASH models. Furthermore, studies have revealed that inhibition of necroptosis via RIPK3 ablation in dietary-related mouse models reduces NASH-related liver inflammation and fibrosis (97, 98). RIPK3 mediates liver injury, inflammation, induction of hepatic progenitor cells/activated cholangiocytes, and liver fibrosis through a pathway suppressed by caspase-8 (99). In addition, in patients with NASH, serum concentrations of RIPK1 and MLKL increase with activity, and RIPK1 inhibition improves NASH features in high-fat diet-fed mice and reverses steatosis via an MLKL-dependent mechanism (100). Mohammed et al. also revealed that increased necroptosis in the liver plays a role in the increased inflammation and fibrosis observed in NASH mice. They found that markers of pro-inflammatory M1 macrophages, NLRP3 inflammasomes, transcript levels of pro-inflammatory cytokines and chemokines such as TNF-α, IL-6, IL-1β, and Ccl2, expression of antioxidant enzymes and heat shock proteins, and markers of fibrosis are significantly increased in NASH mice; however, treatment of NASH mice with a necroptosis inhibitor reverses these conditions (101). In addition, hepatocyte injury or death has been reported to release mitochondrial DAMPs. Mitochondrial DNA, the main active component of mitochondrial DAMPs, can trigger the activation of fibrosis in vivo and in vitro, leading to reduced liver fibrosis. Mitochondrial DNA levels have been found to be significantly elevated in the serum of patients with fibrotic NASH (82).

Significantly higher serum levels of IL-33 have been observed in patients with liver cirrhosis compared to control. A previous study reported that hepatic expression of IL-33 is both required and sufficient for severe hepatic fibrosis in vivo. IL-33 does not mediate fibrosis through direct effects on hepatic stellate cells but rather through IL-33-induced increases in IL-13 production in group 2 innate lymphoid cells and subsequent hepatic stellate cell activation (102). The mechanism of IL-33 release during liver cirrhosis has not been elucidated; however, necroptosis has recently been shown to directly induce the release of nuclear IL-33 in its full-length form (80). Therefore, we propose that IL-33 may be considered a necroptotic DAMP that contributes to liver cirrhosis-related fibrosis.

During age-related liver fibrosis, it is worth noting that necroptosis contributes to an increase in the number of proinflammatory M1 macrophages and expression of proinflammatory cytokines (TNF-α, IL-6, and IL-1β), as well as total collagen content in aging livers. Inhibition of necroptosis by necrostatin-1 (Nec-1) has been shown to reduce elevated collagen deposition in old mice by 2.5-fold to levels similar to those in young mice (3).

Liver RIPK3 and HMGB1 protein expression are correlated with fibrosis stage in patients with chronic hepatitis C virus infection, primary biliary cirrhosis, or alcoholic steatohepatitis (74). Recently, increasing evidence has suggested a role for HMGB1 derived from necroptotic hepatocytes in promoting liver fibrosis (103) and inhibiting the necroptotic pathway-attenuated HMGB1 cytoplasmic translocation and liver damage (104). HMGB1 has been reported to stimulate hepatic stellate cell migration and upregulate type I collagen expression (74). Furthermore, HMGB1 plays an important role in recruiting neutrophils to the injured site of necrotic tissue, resulting in aseptic inflammation, injury amplification, and a reduced survival rate. The S100-calcium-binding protein family, which contains critical DAMP molecules, has also been linked to liver necroinflammation in chronic hepatitis (83). Moreover, necroptosis- and S100A9-dependent NLRP3 inflammasome activation has been reported to induce liver necroinflammation (42). Upregulation of S100A11, a member of the S100 family, significantly contributes to inflammation and fibrosis development in hepatic tissues, which are key drivers of hepatocarcinogenesis (105). In addition, ECM-DAMPs (versican (106), thrombospondin-1 (107), periostin (108), and laminin (109)) promote the local inflammatory immune response and chemokine immune cell recruitment and inflammation, thereby stimulating liver fibrosis (110).

In summary, numerous studies have illustrated that necroptosis produces DAMPs and causes inflammation, eventually leading to fibrosis. Thus, the formation of the necroptosis-DAMP-inflammation axis plays an important role in liver fibrosis.

Aggregation of the ECM protein network is a feature of myocardial fibrosis (111). Myocardial collagen formation is stimulated by oxidative stress and inflammation, leading to fibrosis (112, 113). Myocardial remodeling and fibrosis are caused by several cardiac disorders. Previous studies have revealed that necroptosis is involved in a variety of cardiac disorders. For example, coronary atherosclerosis is one of the leading causes of coronary heart disease (114). Myocardial vascular stenosis and occlusion result from severe atherosclerosis, leading to myocardial ischemia (MI), necrosis, and cardiac remodeling. According to previous studies, expression of RIPK3 and MLKL mRNA increases in atherosclerotic plaques (115), whereas RIPK3 ^KO mice with atherosclerosis exhibit reduced inflammation followed by death (116). In an MI mouse model, RIPK3-dependent necroptosis governs post-ischemic unfavorable remodeling (117). A previous study found that cardiac expression of RIPK3 in MI is upregulated, and RIPK3-deficient animals have a considerably higher ejection fraction and reduced hypertrophy. Ischemia and oxidative stress induce necroptosis, apoptosis, and inflammation in myocardial cells, which leads to myocardial remodeling and heart failure (118).

Myocardial ischemia-reperfusion injury is closely related to necroptosis (118). It has been demonstrated that the RIPK1 inhibitor Nec-1 protects the heart from ischemia-reperfusion injury (119). Myocardial infarction and other ischemic heart diseases lead to cell death, releasing DAMPs such as HMGB1, double-stranded DNA, and ATP (32), activating TLRs and inducing the expression of pro-inflammatory chemokines and cytokines, which result in changes to the ECM (types I and III collagen), fibrosis, and systolic dysfunction (120).

In conclusion, these studies demonstrate that necroptosis plays a significant role in a variety of cardiac disorders. It can limit or alleviate cardiac inflammation and oxidative stress, consequently decreasing cardiomyopathy, excessive ECM synthesis, and fibrosis by blocking major pathway regulatory elements.

Idiopathic pulmonary fibrosis (IPF) is the most prevalent and severe idiopathic non-severe interstitial illness of unknown cause. In IPF, alveolar epithelial cell (AEC) damage and repair failure are followed by fibroblast/myofibroblast activation, fibrosis, and scar formation in the lung parenchyma, leading to lung function loss and respiratory failure (121). RIPK3-mediated necroptosis in AECs contributes to the development of pulmonary fibrosis by DAMP generation, which is part of the IPF etiology (122). RIPK3 expression is higher in IPF lung tissue, particularly in AECs. Bleomycin enhances HMGB1 and IL-1 levels in mouse lung tissue and elevates RIPK3 expression in AECs. Bleomycin-induced lung inflammation and fibrosis have been successfully reduced in RIPK3^KO mice. RIPK3 expression is elevated in the lungs of patients with chronic obstructive pulmonary disease. Nec-1 can reduce inflammation in mice exposed to cigarette smoke (123). Inflammatory cytokines, such as extracellular ATP and DAMPs, have been found to be elevated in the airways of patients with chronic obstructive pulmonary disease (124). Asthma is a common respiratory condition characterized by airway hyperresponsiveness, inflammation, and fluctuating airflow restriction. Eosinophil disintegration and particle release have been linked to airway inflammation. As decreasing RIPK3 expression reduces eosinophil disintegration, necroptosis may be involved in eosinophil death (125). However, RIPK3-deficient animals have been shown to be resistant to allergen-induced asthma (126). Necroptosis is critical for the development of acute lung damage and is indicated by an increase in HMGB1 in the bronchoalveolar lavage fluid of individuals with acute lung damage (127). The concentration of phosphorylated RIPK3 and MLKL increased as the LPS dosage increased in an acute respiratory distress syndrome animal model. Deletion of Nec-1 or RIPK3 genes inhibits necroptosis and improves the lung status of animals with acute respiratory distress syndrome (128). These findings indicate that necroptosis plays a key role in various lung diseases and that DAMPs cause inflammation, which contributes to lung fibrosis.

In the abovementioned severe pulmonary inflammatory diseases, lung epithelial cells and innate immune cells undergoing necroptosis can secrete numerous DAMPs. This triggers the release of proinflammatory cytokines and further aggravates severe inflammation, changing the steady-state lung environment into one with inflammation and amplification of DAMP release, resulting in pulmonary fibrosis. The released DAMPs then bind to TLRs. TLR4 has been shown to promote fibrosis in the lungs (86). HMGB1 may also be a potential target for treating inflammation and improving pulmonary fibrosis. HMGB1 levels are elevated in patients with IPF. The potential pathogenic role of HMGB1 in IPF has been supported by experimental animal models, indicating that anti-HMGB1 antibodies can prevent bleomycin-induced pulmonary fibrosis (72).

After an injury, adipose tissue undergoes a series of different reactions, including cell necrosis, local or systemic inflammatory responses, and complete or incomplete regeneration repair caused by fibrosis or scarring. Adipose tissue fibrosis is difficult to reverse in most cases, posing significant therapeutic challenges and causing considerable discomfort to patients. Adipose tissue fibrosis, similar to other fibrotic disorders, is linked to an increase in ECM storage and synthesis (129, 130).

The most frequently observed ECM proteins in healthy and fibrotic adipose tissues are fibronectin and collagen, respectively. The amount of types I, III, V, and VI collagen is greater in obese and diabetic mice than in normal mice (131). RIPK3 is overexpressed in the white adipose tissue of obese mice fed a choline-deficient high-fat diet. Genetic inactivation of RIPK3 promotes caspase-8-dependent adipocyte apoptosis and increases white adipose tissue inflammation (98). Adipose tissue damage causes changes in adipose cell volume and mass, leading to inflammation, adipose tissue imbalance, and eventually fibrosis (132). Some experiments have also shown that a lack of TLR4 can effectively inhibit the inflammatory response and obesity in adipose tissue (133). Another cause of fat fibrosis is hypoxia. Long-term hypoxia can cause damage, fibrosis, cell aging, and the death of necrotic adipocytes (134).

With advancements in fat transplantation technology, this technique is widely used and recognized. However, due to various factors, this method also has many complications. The most common complications, no/low micrograft retention, infection, oil cysts, and calcification, are associated with graft fat necrosis (135). At present, necrosis of transplanted adipose tissue remains to be further explored, but it is worth noting that complications such as calcification and oil cysts are closely related to fibrosis. Previous studies have shown that necroptosis plays an important role in the occurrence of fibrosis. Therefore, it is reasonable to speculate that necroptosis is related to the formation of fibrosis after fat transplantation. Conversely, following injury, transplanted adipose tissue ruptures some adipocytes, causing intracellular substances to flow out of cells. These can subsequently be used as DAMPs to cause an inflammatory response and aggravate fibrosis. Injured cells in adipose tissue secrete DAMPs such as LPS and fatty acids, activate the TLR4 pathway, and induce an inflammatory response (136). Several proinflammatory chemokines, including TNF-α, inducible nitric oxide synthase, IL-6, IL-8, C-reactive protein, transforming growth factor-β1, soluble intercellular adhesion molecule-1, and monocyte chemoattractant protein-1, are produced following adipocyte death (137–139). M1 macrophages are observed in large numbers in fibrotic adipose tissue. In healthy adipose tissue, permanent macrophages comprise 10–15% of stromal cells (140). The concentration of stromal cells in obese individuals increases by 45%–60% (141, 142). Adipose stromal cells (ASCs) tend to develop into proinflammatory cells in the presence of M1 macrophages. TNF, inducible nitric oxide synthase, and IL-6 are proinflammatory cytokines released by macrophages that trigger this alteration (142, 143). Exposure of ASCs to substances released by M1 macrophages has been shown to enhance ECM remodeling. In addition, ASCs have a proinflammatory profile that causes them to proliferate and migrate while decreasing their differentiation potential (143). This implies that in the presence of the M1 macrophage secretory factor, macrophages induce the formation of profibrotic ASCs. Thus, M1 macrophages play a key role in promoting fat fibrosis. Another study discovered that apoptotic adipocytes can cause fibroblast necroptosis, which promotes collagen production via paracrine pathways (11). In mice, fibrosis has been found to increase following fat transplantation. Macrophages generate macrophage foam cells surrounding apoptotic fat cells or large oil droplets, engulfing oil droplets and other cell debris before necroptosis. Macrophage foam cell necroptosis causes fibrosis by altering the expression of types I and VI collagen in fibroblasts via a paracrine pathway that involves inflammatory cytokines and chemokines.

In addition, ECM proteins are overproduced, and ECM breakdown is inhibited during the development of fibrosis. ECM proteins are produced by various cell types in adipose tissue, including fat progenitor cells, adipocytes, fibroblasts, and myofibroblasts (144). Matrix metalloproteinases and tissue inhibitors of metalloproteinases govern ECM breakdown. Furthermore, matrix metalloproteinases can dissolve ECM components, which can be prevented by some tissue inhibitors of metalloproteinases (145).

Based on current research, necroptosis may be linked to adipose tissue fibrosis, and the essential molecular proteins of the necroptosis pathway could be key to controlling adipose tissue fibrosis. However, further experiments are required to verify this hypothesis.

In summary, our analysis of the relationship between fibrosis and necroptosis in wounds, liver, heart, lung, and adipose tissues illustrated that inhibiting necroptosis can significantly alleviate and improve the degree of fibrosis in tissues and organs. Therefore, in the next section, we review various ways to inhibit necroptosis.