Studies have demonstrated that silicosis begins with inflammation of the lungs. Silicon dioxide (SiO₂) stimulates immune cells such as macrophages, neutrophils (NEUTs), mast cells (MCs), dendritic cells (DCs), T cells, and B cells. Immune cells regulate silicosis- and pulmonary fibrosis-related processes through several molecular pathways. Both the innate and adaptive immune systems are involved in the modulation of silicosis-induced inflammation (Langley et al., 2004; Malaviya et al., 2020).

IL-1β is a downstream product of NLRP3 inflammasome activation and has pro-inflammatory and fibrinogenic effects in silicosis. The excessive secretion of IL-1β can aggravate silicosis, whereas its neutralization or knockout can reverse the progression of the disease (Cavalli et al., 2015; Qin et al., 2021). Studies have shown an association between IL1B gene polymorphism and an increased risk of silicosis (Weng et al., 2015). The importance of IL-1β in silicosis has been demonstrated in both the inflammatory and fibrotic stages. IL-1β can promote inflammatory cell infiltration by stimulating the expression of TNF-α, IL-6, and MCP-1, thereby further amplifying the inflammatory response (Guo et al., 2013; Chen et al., 2014). Moreover, IL-1β contributes to a shift in the Th1/Th2 balance toward a Th2-dominant response during the development of fibrosis (Guo et al., 2013). IL-1β is a pro-fibrogenic cytokine that enhances collagen deposition by increasing the expression of TGF-β (Fan et al., 2001; Guo et al., 2013).

IL-6 is mainly secreted by monocytes, lymphocytes, pulmonary fibroblasts, pulmonary macrophages, and endothelial cells. The levels of IL-6 are significantly increased in the serum and BALF of patients with silicosis (Blanco-Perez et al., 2021). High IL-6 levels exert marked effects on other inflammatory factors, immune cells, and oxidative stress, which are closely related to the pathogenesis of silica-induced pulmonary fibrosis. IL-6 is required for the production of other inflammatory factors, such as IL-1 and TNF-α. The differentiation of CD4⁺ T cells is also modulated by IL-6, which induces the differentiation of Th17 cells but inhibits the production of Tregs. An imbalanced Th17 cell/Treg ratio results in the disruption of the immune response and the promotion of inflammation-related diseases (Kang et al., 2019). In mice, silencing IL-6 receptor-α can ameliorate the secretion of Th2 cytokines (IL-4 and IL-5) and inhibit the activation of the IL-6/JAK/STAT pathway, thereby improving lung function and alleviating the development of silicosis (Tripathi et al., 2010). IL-6 can also influence dust-induced oxidative stress in the lung. The epithelial cells of the lungs produce ROS and express ICAM-1 through an IL-6/AKT/STAT3/NF-κB-dependent pathway, thereby mediating immune cell adherence and infiltration into inflammatory sites and fibrotic lesions (Liu et al., 2018).

IL-17, secreted by macrophages and Th17 lymphocytes, is important for host defenses against extracellular pathogens, and can exacerbate inflammatory responses (Korn et al., 2009). Rats exposed to silica display increased levels of IL-17, which is associated with the progression of fibrosis (Lo Re et al., 2010). The mRNA expression levels of IL-17, retinoid-related orphan nuclear receptor γt (RORγt), and IL-23 are increased in AMs of mice tracheally perfused with silica dust, leading to the local aggregation of Th17 lymphocytes and the production of IL-17. These changes subsequently aggravate the acute inflammatory response in silicosis (Lo Re et al., 2010). IL-23 maintains the phenotype of Th17 lymphocytes, while RORγt is the key transcription factor of Th17 lymphocytes (Korn et al., 2009). The neutralization of IL-17 can reduce neutrophil recruitment and suppress Th17 cell development by decreasing IL-6 and IL-1β content (Hasan et al., 2013). IL-17 modulates the differentiation of Tregs in the early phase of silicosis and promotes the Th1/Th2 immune response, thus influencing silica-induced lung inflammation and fibrosis by regulating the production of IL-22 and IL-1β (Chen et al., 2014; Song et al., 2014). Hence, neutralization of IL-17 with anti-IL-17 antibody could inhibit silica-induced lung inflammation and fibrosis by decreasing Th17 cells and IL-1β, increasing Tregs, and delaying silica-induced Th1/Th2 immune response (Chen et al., 2014).

Other interleukins, such as IL-13, IL-18, and IL-8, also promote fibrosis through a variety of mechanisms, while cytokines such as IL-7, IL-12, and IL-22 can inhibit the formation of fibrosis by reducing collagen deposition (She et al., 2021).

TNF-α, produced by activated macrophages, fibroblasts, and lymphocytes, can initiate a complex cascade of inflammatory responses in the early inflammatory stage and during the late development of silica-induced fibrosis (Park et al., 2012). TNF-α increases the risk of pulmonary fibrosis by inducing the expression of IL-1, IL-6, and IL-8. Meanwhile, TNF-α can stimulate the expression of TGF-β1 through the ERK signaling pathway (Xie et al., 2021), which promotes fibrosis progression by inducing ECM synthesis and deposition in fibroblasts (Morrison and Davey, 2009; Jiang et al., 2015). That TNF-α plays an important role in the development of silicosis has been demonstrated in SiO₂-treated rats. After a course of infliximab (anti-TNF-α antibody) injections, the number of inflammatory cells was decreased, collagen deposition was reduced, NF-κB signaling was inhibited, and iNOS expression was downregulated, leading eventually to an improvement in pulmonary function (Zhang et al., 2018). Silicosis can be prevented with the administration of an anti-TNF antibody but is aggravated by exogenous recombinant TNF-α application. The latter augments fibroblast growth, collagen deposition, and cell necrosis (Piguet, 1990). Moreover, in silicosis, TNF-α stimulation promotes the secretion of other cytokines, influences the function of signaling pathways, and potentiates the proliferation of fibroblasts and the deposition of collagen. Infliximab is the anti-TNF-α antibody, which improve silica-induced pulmonary inflammation by decreasing the TNF-α, inhibiting NF-κB signaling as well as oxidant status (iNOS) (Zhang et al., 2018).

TGF-β is a well-characterized fibrogenic mediator and it has long been known that TGF-β expression is increased in silicosis. In rats, after a single intratracheal infusion of silica, TGF-β peptide was detected in fibroblasts and macrophages surrounding silicotic granuloma and fibroblasts adjacent to hyperplastic type II cells in the lungs (Williams et al., 1993). The major role of TGF-β in silicosis is the promotion of ECM accumulation, EMT induction, and the inhibition of matrix degradation. During pulmonary fibrosis, the TGF-β/Smad signaling pathway is activated, which increases the expression of collagen and fibronectin genes. TGF-β binds to TGF-β receptors (TβR) on lung fibroblasts, leading to the phosphorylation of Smad2/3. Phosphorylated Smad2/3 then translocate to the nucleus and induce the expression of genes that promote EMT and the transformation of fibroblasts into myofibroblasts, resulting in the production of a large amount of ECM (Attisano and Wrana, 2002; Zhang et al., 2021). An uncontrolled, continued EMT may result in fibrosis that has a close relationship with silicosis (Stone et al., 2016). TGF-β can also inhibit matrix degradation by decreasing the secretion of proteases and increasing that of protease inhibitors such as MMPs and tissue inhibitor of metal protease (TIMP) (Yang et al., 2011). Through these various means, TGF-β can cause pulmonary fibrosis by promoting the accumulation of a large amount of ECM in the lungs. TGF-β can also promote pulmonary fibrosis via the activation of the ERK and PI3K/AKT pathways (Kolahian et al., 2016). Hence, as a key inflammatory factor in silicosis, any way to downregulate the expression of TGF-β or to neutralize secreted TGF-β might be possible to delay the progression of silicosis. According to this, the anti-TGF-β antibody HTPEP-001 was developed and is currently in clinical phase I (Liu et al., 2023).

Many growth factors, such as PDGF, fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), and connective tissue growth factor (CTGF), can promote fibrosis (Jiao et al., 2021; Zhang et al., 2021). The role of PDGF in fibrosis has been particularly well-studied. It has been shown that SiO₂ can induce PDGF secretion in Tregs, thereby stimulating fibroblasts and promoting pulmonary fibrosis (Lo Re et al., 2011). The overexpression of PDGF aggravates silicosis by increasing Ca²⁺ release through the PI3K signaling pathway, ultimately influencing the expression of ECM-related genes in pulmonary fibroblasts (Mukherjee et al., 2013).

CTGF, the most direct downstream effector of TGF-β, influences the pathogenesis of silicosis by promoting the synthesis of ECM, the proliferation and migration of fibroblasts, and EMT (Paradis et al., 1999; Jiao et al., 2021). In silicosis, the TGF-β/CTGF pathway enhances inflammation and fibrosis primarily by interacting with Smad or MAPK (Jiao et al., 2021). A high concentration of CTGF is detected in fibroblasts treated with the supernatant of SiO₂-stimulated AMs. However, anti-CTGF antibody administration prevents CTGF from activating downstream pro-fibrotic signaling, thereby attenuating silicosis (Cui et al., 2018). Combined, these observations indicate that CTGF exerts pro-fibrotic effects in silicosis.

VEGF plays a key role in lymphangiogenesis, which is an important channel for the removal of dust and inflammatory mediators in the early stage of silicosis (Yu et al., 2016). Jinsong et al. found that VEGF expression levels are higher in BALF and AMs of patients with silicosis than in healthy controls. In lymphatic endothelial cells cultured in supernatant medium of silica-treated human monocyte-macrophages (U937 cells), VEGF can promote the expression of VEGF receptor 3 (VEGFR3) and lymphatic vessel endothelial hyaluronic acid receptor 1 (LYVE-1), which can influence lymphangiogenesis by upregulating the Src/eNOS signaling pathway (Zhang et al., 2021). Specifically, VEGF stimulates lymphangiogenesis in silicosis, which promotes inflammation, oxidative damage, and, ultimately, fibrosis.

MCP-1 (also known as CCL2), mainly derived from fibroblasts and macrophages, is a member of the C-C class chemokine family and a key factor in the initiation of inflammation (Deshmane et al., 2009; Melgarejo et al., 2009). MCP-1 can regulate the migration, chemotaxis, and recruitment of macrophages and T lymphocytes during various types of inflammation (Lee and Song, 2017). Boitelle A et al. found that coal workers have higher levels of MCP-1 compared with healthy controls. They speculated that MCP-1 promotes the migration of peripheral blood mononuclear cells out of blood vessels into tissues, causing mononuclear macrophages to accumulate in the alveolar lumen, and thus influencing the occurrence of alveolitis in coal worker’s pneumoconiosis (Boitelle et al., 1997). When exposed to SiO₂, human pulmonary fibroblasts (HPF-α) release MCP-1. The increased release of MCP-1 and its receptor, CCR2, promotes cell activation and migration (Liu et al., 2015).

Inflammatory factors play important roles in silicosis, the interaction between them is complicated. However, regulation of some of these factors suggest a strategy to mitigate the progression of fibrosis in silicosis, and some drugs targeting on these factors have revealed good therapeutic potential (Table 1).

NF-κB is a nuclear transcription factor with a key role in pro-inflammatory signaling and represents a major regulatory node in a complex inflammatory signaling network (Lawrence, 2009; Rius-Pérez et al., 2020). NF-κB is rapidly and persistently activated in acute and chronic inflammatory disorders and plays a critical role in host defenses against pathogens and toxic substances, such as silica (Kang et al., 2000; Zhang et al., 2017; Dorrington and Fraser, 2019). Following exposure to silica, NF-κB is rapidly released from IκB, following which it translocates into the nucleus and initiates the transcription of specific genes, such as TNF, IL1B, and IL6, which, in turn, can further aggravate the inflammatory response and fibrosis (Rojanasakul et al., 1999; Porter et al., 2002). The systemic blockage of NF-κB activation can markedly ameliorate SiO₂-induced inflammatory responses, collagen deposition, apoptosis, and fibrosis in mice. However, in transgenic mice with lung epithelial cell-specific inhibition of NF-κB activation, lung inflammation is decreased following silica exposure, whereas collagen deposition and apoptosis levels are increased (Di Giuseppe et al., 2009). As mentioned earlier, silica exposure induces NF-κB activation in macrophages, which modulates macrophage apoptosis. To clarify the role of NF-κB activation in silica-induced apoptosis, macrophages were treated with BAY 11-7085, a powerful inhibitor of IκB phosphorylation. The absence of NF-κB was found to increase silica-induced apoptosis in macrophages (Gambelli et al., 2004). The absence of NF-κB was found to increase silica-induced apoptosis in macrophages. Meanwhile, whereas the systemic inhibition of NF-κB protects against silica-induced lung injury, specific NF-κB inhibition in macrophages and lung epithelial cells appears to promote cell apoptosis and aggravate silicosis.

The generic MAPK signaling pathway is shared by four distinct cascades, namely, ERK1/2, JNK1/2/3, p38-MAPK, and ERK5. The MAPK signaling pathway is the primary regulatory module of various cellular processes, such as cell proliferation, differentiation, and responses to stress. ROS, cytokines, and other substances produced following exposure to SiO₂ act on MAPK/ERK pathway-related receptors and activate downstream signals, thereby promoting silicosis formation and development (Ding et al., 1999). Studies have indicated that the SiO₂-induced activation of fibroblast proliferation, ECM production, inflammatory responses, and apoptosis is mediated by the MAPK/ERK signal pathway, at least partly. The MAPK/ERK inhibitor (PD98059) exerts a marked antagonistic effect on silica-induced lung inflammation by inhibiting the expression of TNF-α and TGF-β (Li et al., 2009). Silica exposure induces ERK1/2 phosphorylation, which can protect against macrophage apoptosis, in contrast to that observed with PD98059 treatment (Gambelli et al., 2004). Silica induces early growth response protein 1 (EGR-1) activation via the MAPK/ERK pathway, resulting in the modulation of the expression of inflammation-related genes (PDGF, ICAM-1) and fibrosis-related genes that affect matrix balance (MMP1, fibronectin), which promotes the inflammatory response, fibroblast proliferation, and ECM synthesis (Zeng et al., 2005).

p38-MAPK, a central mediator of inflammatory and stress responses, is a major cellular signal transducer of extracellular stress signals induced by lipopolysaccharide (LPS), endotoxins, and pro-inflammatory cytokines (Yeung et al., 2018). p38-MAPK is activated by silica, following which it phosphorylates an array of substrates in both the cytoplasm and nucleus, thereby influencing inflammation, cell differentiation, and cell growth (Wang et al., 2016). Ample evidence suggests that p38-MAPK regulates signals involved in the development of fibrosis by mediating EMT and promoting TGF-β secretion. Treatment with SB203580, a p38 kinase inhibitor, can significantly reduce silica-induced TGF-β secretion and EMT, as evidenced by the consequent induction of E-cadherin and repression of vimentin and α-SMA. p38 inhibitor ameliorates silica-induced pulmonary fibrosis, which may be related to the inhibition of ZEB-1, ZEB-2, and Twist expression (Wang et al., 2016).

SiO₂ is also known to activate JNK in vivo and in vitro. The JNK inhibitor BI-78D3 not only completely blocks the production of leukotriene B₄, IL-1β, and CXC chemokines in macrophages, MCs, and neutrophils, but also reduces SiO₂-induced sterile inflammation in an air-pouch model in mice (Hegde et al., 2018).

Pro-apoptotic Fas and FasL play critical roles in pulmonary immune homeostasis, immune surveillance, and autoimmunity involving T cells. Clinical research has shown that patients with silicosis exhibit elevated levels of soluble Fas in serum and peripheral blood mononuclear cells, as well as higher levels of other FAS transcript variants and reduced membrane Fas expression in lymphocytes (Otsuki et al., 2006). Fas/FasL signaling aggravates inflammation-related apoptosis in epithelial cells and AMs, which consequently release IL-1β and chemokines, leading to neutrophil infiltration and lung injury (Dosreis et al., 2004; Hoffmeyer et al., 2010). Patients with silicosis administered an anti-FasL antibody exhibit reduced levels of the pro-apoptotic factors Fas and caspase-3 and the inflammatory mediators TGF-β and IL-8 (Yao et al., 2013). Fas ligand-deficient GLD mice instilled with silica do not develop silicosis. These animals display significantly reduced neutrophil extravasation into the bronchoalveolar space, decreased TNF-α production, reduced pulmonary inflammation, and macrophage apoptosis (Borges et al., 2001). In wild-type mice administered an anti-FasL monoclonal antibody, neutrophil accumulation in the lung parenchyma is inhibited, as is macrophage apoptosis, and even silicotic fibrosis (Borges et al., 2001). The Fas/FasL pathway plays an important role in regulating the levels of inflammatory cytokines and promoting lung inflammation and fibrosis.

The cGAS-STING signaling pathway has emerged as an essential mediator of inflammation in infection, cellular stress, and tissue damage (Decout et al., 2021). Cyclic GMP-AMP synthase (cGAS) is a recently identified intracellular pattern recognition receptor (PRR) that can recognize abnormal cytoplasmic dsDNA, following which it catalyzes the synthesis of cyclic GMP-AMP (cGAMP). cGAMP then activates stimulator of interferon genes (STING), which promotes the secretion of type-I IFN and other inflammatory factors, thereby influencing the immune response (Motwani et al., 2019). The cGAS-STING pathway is essential for silica-induced lung inflammation. Activation of the STING pathway by airway silica leads to cell death, self-dsDNA release, an increase in ROS generation, and STING/type-I-IFN-dependent acute lung inflammation (Messaoud-Nacer et al., 2022). cGAS recognizes leaked dsDNA and activates STING in vivo after silica exposure. When DNase I is used to degrade dsDNA, silica-induced STING activation and the downstream type-I-IFN response are inhibited (Benmerzoug et al., 2018).

Ample evidence supports that SiO₂ activates inflammatory cytokines such as IL-1β, IL-6, TGF-β, TNF-α, growth factors, and CCL and promotes pulmonary fibrosis (Boitelle et al., 1997; Blanco-Perez et al., 2021), suggesting that increased inflammatory cytokine levels may be used as typical biomarkers for the clinical diagnosis of silicosis.

HO-1, an inducible antioxidant stress protein, is one of the most important downstream regulatory effectors of nuclear factor-erythroid 2 related factor 2 (Nrf2) and exhibits anti-inflammatory and antioxidant properties (Loboda et al., 2016; Zhang et al., 2021). HO-1 is a rate-limiting enzyme in heme catabolism, degrading heme to carbon monoxide (CO), biliverdin (BV), and ferrous ion (Fe²⁺) (Ayer et al., 2016). It has been suggested that all these byproducts are important contributors to the anti-inflammatory activity of HO-1. The endogenous mediator CO inhibits the expression of pro-inflammatory cytokines, such as TNF-α and IL-1β, while simultaneously increasing the expression of the anti-inflammatory cytokine IL-10 (Abraham and Kappas, 2008). It is reported that the HO-1/CO axis regulates inflammation and the immune system (Ryter, 2020). BV and its downstream reductive derivative bilirubin (BR) are potent anti-inflammatory factors that can inhibit C5aR expression in macrophages and reduce pro-inflammatory cytokine expression through mTOR signaling (Bisht et al., 2014). HO-1 is present in silicotic nodules, where it exerts protective effects by attenuating lung inflammation (Nakashima et al., 2018), suppressing ROS activity and subsequent inflammatory and pathologic changes, and thereby attenuating disease progression (Sato et al., 2006; Nakashima et al., 2018). Low serum concentrations of HO-1 can predict impaired lung function in chronic silicosis. These observations imply that HO-1 is a key biomarker suitable for the monitoring of at-risk patients and a potential therapeutic target for silicosis (Sato et al., 2006; Sato et al., 2012).

The nuclear factor high-mobility group box 1 protein (HMGB1), which is actively secreted by inflammatory cells or passively released from necrotic cells, was recently found to function as an important late inflammatory mediator (Ciucci et al., 2011). High levels of HMGB1 have been strongly linked to silicosis; each 1 ng/mL increase in plasma HMGB1 levels is positively correlated with an increased risk of silicosis (Ma et al., 2018). Jixuan et al. demonstrated that the overexpression of HMGB1 induced by SiO₂ exposure can influence the progression of silicosis, including pulmonary inflammation and fibrosis, via EMT.

The authors found that the neutralizing antibody-mediated abrogation of HMGB1 attenuated silica-induced lung inflammation, fibrosis, and EMT, whereas the application of recombinant HMGB1 exerted the opposite effect (Ma et al., 2020). HMGB1 acts to alert cells to hazardous surroundings, such as in the presence of silica. The increased secretion of HMGB1 can strongly promote leukocyte recruitment and activation, which triggers tissue repair and aggravates silicosis (Rabolli et al., 2014; Yang et al., 2020). Accordingly, there is sufficient evidence to show that HMGB-1 is involved in the progression of silicosis, suggesting that it may represent a potential diagnostic biomarker and therapeutic target for silicosis.

The NLRP3 inflammasome is a large multi-protein complex composed of the core protein NLRP3, apoptosis-associated speck-like protein containing a CARD (caspase activation and recruitment domain) (ASC), and pro-caspase-1 (He et al., 2016). Once activated, caspase-1 cleaves inactive IL-1β and IL-18 into their mature forms, which generates pro-inflammatory effects (Jessop et al., 2017). Abundant evidence suggests that NLRP3-mediated inflammation plays an essential role in the fibrogenesis and pathogenesis of silicosis (Cassel et al., 2008; Peeters et al., 2014). NLRP3 inflammasomes in AMs are major inducers of cytokine secretion. The phagocytosis of SiO₂ particles activates NLRP3 inflammasomes via four different mechanisms, namely: 1) SRs on the surface of AMs recognize and induce the internalization of SiO₂ particles, leading to NLRP3 inflammasome activation (Hari et al., 2014); 2) AMs phagocytose SiO₂, resulting in stress injury and the release of intracellular ATP to the outside of the cell. P2X7 receptors (P2X7R; ATP-gated ion channels) on the cell membrane recognize ATP, leading to the opening of some of the channels and the subsequent activation of NLRP3 by K⁺ outflow and Na⁺ influx (Luna-Gomes et al., 2015); 3) engulfed SiO₂ particles promote the generation of a large amount of ROS, resulting in NLRP3 activation (Harijith et al., 2014); and 4), the contents released following lysosomal rupture activate the NLRP3 inflammasome. When activated, the NLRP3 inflammasome, via activated caspase-1, converts pro-IL-1β and pro-IL-8 to their active forms. IL-1β and IL-8 then act to increase the production of other pro-inflammatory and pro-fibrotic cytokines, which promotes fibrosis (Jessop et al., 2017).

Normal macrophages produce large amounts of IL-1β when exposed to silica crystals. However, ASC- and NLRP3-deficient mice both exhibit reduced inflammation and granuloma formation (Cassel et al., 2008). Macrophages lacking NLRP3 or ASC do not release cleaved IL-1β in response to silica crystal exposure (Hornung et al., 2008). Together, these findings indicate that the NLRP3 inflammasome plays a central regulatory role in silicosis, and further suggest that activated NLRP3 may serve as an important biomarker of silicosis.

Dust inhalation can induce oxidative stress responses, leading to the generation of a large amount of ROS, reactive nitrogen species (RNS), lipid peroxides, and other free radicals, all of which can damage mitochondrial DNA and increase the incidence of DNA methylation (Fubini and Hubbard, 2003; Zhang et al., 2019). Aberrant DNA methylation profiles have been found in the lung tissues of patients with silicosis. Namely, 86,770 and 79,660 CpG sites were found to differ significantly in methylation status between the lungs of patients with early-stage and advanced-stage disease, respectively, and those of healthy individuals (Zhang et al., 2016). The DNA of a large number of genes is abnormally methylated in SiO₂-treated lung fibroblasts. Methylated DNA immunoprecipitation (MeDIP) experiments showed that these genes are mainly involved in the transformation and differentiation of fibroblasts (Li et al., 2017). DNA methylation is also found in genes associated with the WNT signaling pathway, which contributes to proliferative, fibrogenic, and inflammatory responses to silica exposure in lung epithelial cells (Perkins et al., 2016). These findings suggest that specific DNA methylation may have value as a diagnostic biomarker for silicosis.