Context-dependent roles of palmitoylation in acute respiratory distress syndrome: integrating inflammation, cell death and repair

Abstract

Acute respiratory distress syndrome (ARDS) is a life-threatening condition characterised by dysregulated inflammation, immune imbalance and impaired alveolar repair. Despite advances in supportive care, effective targeted therapies remain limited. Palmitoylation, a reversible lipid-based post-translational modification, has recently emerged as a regulatory mechanism in ARDS pathogenesis. Acting in a context-dependent manner, palmitoylation affects key processes, including immune activation, programmed cell death and epithelial remodelling. Accumulating evidence suggests that palmitoylation may exert dual roles in ARDS: it can promote inflammation and immune evasion in the early phase, while contributing to resolution and tissue repair during later stages. This review summarises current findings regarding the spatial and temporal regulation of palmitoylation in immune and structural cells involved in ARDS, including its effects on inflammasome activation, epithelial–immune interactions and fibrotic progression. Therapeutic approaches under investigation include selective inhibition of palmitoyltransferases (zinc finger aspartate-histidine-histidine-cysteine motif-containing-type palmitoyltransferase family), modulation of depalmitoylation enzymes and substrate-targeted strategies. Several preclinical studies support the feasibility of targeting palmitoylation to reduce lung injury and improve immune regulation. Overall, palmitoylation represents a potential regulatory node in ARDS pathophysiology. Further research is required to clarify its cell-specific functions and to assess the translational potential of palmitoylation-based interventions.

Shareable abstract

Palmitoylation dynamically regulates inflammation, pyroptosis and tissue repair in ARDS. Its context-dependent roles offer novel insights and therapeutic opportunities for modulating immune responses and restoring alveolar homeostasis. https://bit.ly/3I9bbFr

Introduction

Acute respiratory distress syndrome (ARDS) is a severe and acute lung injury characterised by diffuse damage to the alveolar–capillary barrier, resulting in pulmonary oedema, impaired gas exchange and systemic inflammatory response [1]. The pathogenesis of ARDS is highly complex, involving multiple cell types and molecular signalling pathways, including inflammatory responses, oxidative stress and excessive immune activation [2, 3]. Currently, the primary treatment strategies for ARDS rely on supportive care, such as mechanical ventilation and fluid management, and significant therapeutic advancements remain limited.

Palmitoylation is a reversible lipid modification in which a palmitic acid (C16:0) moiety is covalently attached to proteins, typically at cysteine residues [4]. Palmitic acid, a saturated fatty acid, is widely distributed in biological systems, particularly within cell membranes. Palmitate is synthesised from fatty acids via fatty acid synthase (FASN), a process initiated by glucose uptake in hepatocytes, in which glycolysis converts glucose into pyruvate [5]. Pyruvate dehydrogenase then catalyses the conversion of pyruvate into acetyl-coenzyme A (CoA) within mitochondria, which is subsequently carboxylated by acetyl-CoA carboxylase to form malonyl-CoA. Through a multistep catalytic reaction, FASN facilitates the synthesis of palmitate from acetyl-CoA and malonyl-CoA [6, 7]. Palmitoylation is primarily an enzyme-mediated process, catalysed by a family of palmitoyltransferases, which share a conserved aspartate-histidine-histidine-cysteine (DHHC) motif within their cysteine-rich domains [8–10]. Palmitoylation is a highly conserved post-translational modification present in all eukaryotic organisms, playing a critical role in regulating protein stability, subcellular localisation, membrane trafficking, interactions with effector proteins, enzymatic activity and various other cellular processes [11, 12]. This modification has profound effects on immune regulation and cellular function, particularly in inflammatory responses, oxidative stress and tissue repair [13]. In lung injury, the regulation of palmitoylation modulates immune cell function, enhances tissue repair capacity and mitigates oxidative damage, which may be pivotal for restoring pulmonary function. In contrast, depalmitoylation involves the removal of palmitate moieties from proteins by depalmitoylating enzymes such as α/β hydrolase domain-containing protein 17A (ABHD17A) and palmitoyl-protein thioesterase 1 (PPT1) [14–16], thereby altering protein membrane localisation and function [17]. Depalmitoylation is often associated with the suppression or inhibition of specific signalling pathways, making it crucial for controlling excessive immune and inflammatory responses. It plays a vital role in restoring immune homeostasis, reducing immune hyperactivation and facilitating tissue repair [18].

The core of ARDS pathology is an imbalance between uncontrolled inflammation and tissue repair, and palmitoylation serves as a key node connecting innate immune overactivation with lung injury repair by dynamically modulating immune receptor localisation, inflammatory vesicle activity and cell death programmes. However, the paradoxical roles of palmitoylation in pro-inflammatory and anti-inflammatory, pro-death and pro-survival responses make its therapeutic application in ARDS particularly challenging. Rather than functioning as a static membrane-anchoring signal, palmitoylation is dynamically regulated by DHHC enzyme activity, substrate availability and membrane microenvironment. This modification integrates into key signalling cascades, such as G protein-coupled receptor, Wnt/β-catenin and Ras/mitogen-activated protein kinase (MAPK), where it fine-tunes protein localisation, stability and activation [19–21]. Given the stage- and subtype-specific immune landscapes in ARDS, decoding the context-dependent regulatory functions of palmitoylation is essential for developing precise therapeutic strategies.

In this review, we systematically summarise the molecular details of the regulatory network of palmitoylation and explore how to balance immune defence and pathological injury by precisely intervening in this network.

We performed a comprehensive literature search using PubMed, Embase and Web of Science to identify relevant studies on protein palmitoylation and ARDS. Emphasis was placed on peer-reviewed articles published in the past 5 years, with no strict time restriction. Studies involving pulmonary inflammation, immune regulation and lipid modification mechanisms were prioritised. Additional references were identified through manual screening of cited articles.

Regulation of immune cells by palmitoylation

The immune landscape of ARDS is shaped by a complex interplay of innate and adaptive cell populations whose activation, differentiation and effector functions are tightly regulated at the post-translational level [22]. Among these modifications, palmitoylation, a reversible lipid-based acylation of cysteine residues, has emerged as a context-dependent modulator of immune signalling pathways. By controlling the membrane localisation, stability and protein–protein interactions of key signalling molecules, palmitoylation fine-tunes immune responses in a cell type-specific manner. Recent studies have identified distinct zinc finger DHHC (zDHHC) family palmitoyltransferases that modulate inflammatory cascades in neutrophils, macrophage polarisation, antigen presentation by dendritic cells (DCs) and immunosuppressive checkpoint expression in T-cells. This section summarises the emerging evidence for palmitoylation-mediated regulation of immune cell function in ARDS, highlighting its potential as a therapeutic target to restore immune homeostasis (figure 1).

FIGURE 1.

Immune cell-specific regulation by palmitoylation in acute respiratory distress syndrome (ARDS) immunoregulation. Palmitoylation of key signalling proteins by zinc finger aspartate-histidine-histidine-cysteine motif-containing (zDHHC) family members modulates neutrophil activation, macrophage polarisation, regulatory T-cell (Treg)-mediated tolerance, dendritic cell antiviral signalling and B-cell antigen responses, contributing to immune dysregulation or resolution during lung injury. ASC: apoptosis-associated speck-like protein containing a CARD; BCR: B-cell receptor; CD: cluster of differentiation; Foxp3: forkhead box protein P3; cGAS: cyclic GMP-AMP synthase; CMA: chaperone-mediated autophagy; GSDMD: gasdermin D; HSC70/LAMP2A: heat shock cognate protein 70/lysosome-associated membrane protein 2A; IL-1β: interleukin 1β; IRF3: interferon regulatory factor 3; MYD88: myeloid differentiation primary response 88; NF-κB: nuclear factor κB; NLRP3: nucleotide-binding oligomerisation domain, leucine-rich repeat, and pyrin domain-containing 3; PD-L1: programmed death ligand 1; STING: stimulator of interferon gene; TBK1: TANK-binding kinase 1; TCR: T-cell receptor; TGN: trans-Golgi network; TLR4: Toll-like receptor 4; TNF-ɑ: tumour necrosis factor ɑ.

Neutrophils

Neutrophil chemotaxis depends critically on the palmitoylation of chemokine receptors such as CXCR1/2, mediated by specific zDHHC enzymes. This lipid modification promotes receptor partitioning into lipid rafts, enhancing Gi protein-driven activation of phosphoinositide 3-kinase-γ (PI3Kγ) and Rac GTPases, which orchestrate F-actin polarisation and directional migration. Pharmacological inhibition of palmitoylation using 2-bromopalmitate (2-BP) disrupts cytoskeletal polarisation and impairs neutrophil migration, paralleling its barrier-protective effect in endothelial cells, where 2-BP attenuates pyruvate kinase M2 (PKM2) palmitoylation and mitigates dysfunction [23]. Conversely, blocking depalmitoylation sustains receptor palmitoylation and prolongs inflammatory signalling, leading to non-resolving neutrophil infiltration in chronic inflammation [24].

Additionally, palmitoylation modulates neutrophil function by modifying surface receptors, such as integrins and Toll-like receptors (TLRs), thereby amplifying neutrophil responses to chemokines. Neutrophils with enhanced palmitoylation modifications exhibit stronger chemotactic behaviour and antimicrobial activity, contributing to the early phase of immune responses [25].

Furthermore, palmitoylation is also involved in the regulation of inflammatory cytokine release by neutrophils. In both in vitro and animal studies, Kim et al. [26] demonstrated that blocking myeloid differentiation primary response 88 (MyD88) palmitoylation releases its brake on chemotaxis, thereby improving directional migration toward infection sites, and improves survival in septic mice. Their findings revealed that zDHHC6-mediated palmitoylation of MyD88 at cysteine 113 (Cys113), a key adaptor protein in TLR signalling, facilitates the activation of inflammatory pathways, allowing neutrophils to sense pathogens and trigger downstream immune responses. More specifically, zDHHC6-mediated palmitoylation of MyD88 enhances TLR complex assembly, promoting pathogen sensing. While this process is essential for immune defence under physiological conditions, excessive immune activation and cytokine release in diseases such as acute lung injury (ALI) can exacerbate alveolar–capillary barrier damage, leading to pulmonary oedema and impaired gas exchange [26]. Thus, although palmitoylation enhances immune responses, its dysregulation may contribute to tissue damage under pathological conditions. Modulating palmitoylation levels in neutrophils may represent a potential strategy to control cytokine release and mitigate excessive inflammation-induced lung injury.

Macrophages

Macrophages are key immune cells in pulmonary immune responses, playing essential roles in inflammation, immune tolerance and tissue repair. Their function in acute and chronic inflammatory responses is highly complex, because they can adopt either a pro-inflammatory M1 phenotype, which amplifies immune responses, or an anti-inflammatory M2 phenotype, which promotes tissue repair and immune tolerance [27].

Macrophage polarisation (M1/M2) shapes their functional roles in inflammation and tissue repair. Recent studies reveal that site-specific palmitoylation of nucleotide-binding oligomerisation domain, leucine-rich repeat, and pyrin domain-containing 3 (NLRP3) critically regulates inflammasome activation in macrophages. ZDHHC5 catalyses palmitoylation at Cys130, enhancing NLRP3 oligomerisation and facilitating its interaction with NIMA-related kinase 7 (NEK7), which promotes apoptosis-associated speck-like protein containing a CARD (ASC) speck formation, caspase-1 activation and interleukin (IL)-1β release [28]. In contrast, ZDHHC12 mediates palmitoylation, which recruits heat shock cognate protein 70 (HSC70) and lysosome-associated membrane protein 2A (LAMP2A) to initiate chaperone-mediated autophagy, thereby degrading NLRP3 and limiting prolonged inflammation [29, 30]. ZDHHC7 also palmitoylates NLRP3 at Cys126 to promote its localisation to the resting trans-Golgi network and ASC recruitment, while the ZDHHC7/APT2 complex can depalmitoylate NLRP3 to prevent excessive inflammation. This dual regulation provides a temporal checkpoint between pro-inflammatory activation and immune resolution during lung injury [31].

T-cells

T-cells are central to adaptive immune responses, particularly in chronic immune regulation and immune tolerance. Studies have shown that palmitoylation modulates T-cell activation by regulating the palmitoylation of the T-cell receptor (TCR) and co-stimulatory molecules, thereby influencing T-cell function. Palmitoylation enhances TCR signalling efficiency, promoting T-cell activation and proliferation. Several key molecules involved in TCR signal transduction undergo palmitoylation, including Src family kinases lymphocyte-specific protein tyrosine kinase (LCK) and FYN, the transmembrane adaptor linker for activation of T-cells, and the co-receptors CD4 and CD8 [32, 33]. These palmitoylation modifications are crucial for the functional organisation of TCR signalling complexes, ensuring efficient signal transmission. Src family kinases, which undergo palmitoylation, play a key role in adaptive immunity by initiating signalling cascades upon interaction with lipid raft-associated immune receptors in the plasma membrane [28]. This process is essential for T-cell activation, differentiation and immune response modulation, further highlighting the importance of palmitoylation in T-cell-mediated immunity. Palmitoylation of Src family kinase Lck at cysteine residues Cys3 and Cys5 facilitates its membrane association. Further studies have demonstrated that site-specific palmitoylation at Cys3 is particularly crucial for LCK localisation to lipid rafts [34]. Similarly, palmitoylation of FYN, another Src family kinase involved in T-cell signal transduction, plays a significant role in its membrane attachment [35]. FYN undergoes palmitoylation at Cys3 and Cys6 as the primary site essential for lipid raft association. Within lipid rafts, activated LCK phosphorylates FYN, thereby initiating TCR/CD3 activation and downstream signalling cascades. Several DHHC palmitoyltransferases have been identified as mediators of FYN palmitoylation [36].

In ARDS, excessive activation of T-cells can exacerbate immune responses, leading to excessive cytokine release and tissue damage. Depalmitoylation plays a crucial role in maintaining T-cell immune tolerance. By removing palmitoylation modifications, depalmitoylation prevents excessive T-cell activation, thereby protecting self-tissues from immune attack [37]. This mechanism is particularly critical in chronic immune responses and autoimmune diseases. For instance, palmitoylation of programmed death ligand 1 (PD-L1) at Cys272, mediated by zDHHC3, stabilises its membrane localisation and prevents lysosomal degradation, thereby sustaining immune checkpoint signalling and promoting immune evasion in inflamed lung tissue [38]. In lung injury, the establishment of immune tolerance is essential to prevent the immune system from attacking self-tissues. Palmitoylation may contribute to reducing autoimmune responses by regulating T-cell immune tolerance. Some studies suggest that palmitoylation influences the function of regulatory T-cells (Tregs), promoting their role in immune tolerance, which in turn mitigates excessive immune responses and protects lung tissue from immune-mediated damage.

Recent studies have shown that the activity of forkhead box P3 (Foxp3) is regulated by palmitoylation. Foxp3 palmitoylation, catalysed by DHHC2, enhances Treg function and immune tolerance, potentially reducing chronic inflammation and fibrosis [39].

B-cells

B-cells primarily contribute to immune defence by producing antibodies against foreign pathogens. Although their role in the immune response to lung injury is not as prominent as that of T-cells and macrophages, B-cells are still involved in immune regulation and the establishment of immunological memory [40, 41].

B-cells are key components of the adaptive immune response, responsible for antigen recognition and the initiation of immune responses. During immune activation, B-cells bind to antigens through their surface immunoglobulin receptor, the B-cell receptor (BCR), leading to activation. Palmitoylation of BCR and its co-receptor complex influence B-cell activation and immune responses. This modification not only enhances B-cell antigen recognition but also facilitates rapid immune responses, particularly in processes such as antigen recognition, cell proliferation, differentiation and antibody production. The interaction between BCR and the co-receptor complex amplifies and prolongs signalling, while the stabilisation of lipid rafts further enhances signal transduction [42]. This stable signalling cascade regulates B-cell immune responses by modulating receptor function through palmitoylation, thereby promoting B-cell activation, immune factor secretion and memory cell formation. Palmitoylation also modulates metabolic adaptions to support plasma cell differentiation. Conversely, in chronic lung injury, dysregulated extrafollicular B-cell activation drives autoantibody production, highlighting context-dependent roles. Furthermore, a key aspect of immune regulation involves the aggregation of a stable BCR complex within lipid rafts via palmitoylation, which facilitates precise modulation of B-cell immune responses. Immune tolerance during immune activation is another crucial regulatory mechanism, and palmitoylation contributes to preventing excessive immune activation and autoimmune responses by modulating BCR signalling. During the formation of immune memory, the impact of palmitoylation on B-cells is particularly significant. By stabilising the BCR complex, palmitoylation ensures the functionality of memory B-cells, allowing them to rapidly activate and produce antibodies upon re-exposure to the same antigen. This mechanism is essential for vaccine immunology and long-term protection against infections. A recent study identified novel palmitoylated proteins in B lymphocytes, with CD20 receiving notable attention and validation [43]. These findings suggest that palmitoylation not only regulates the BCR complex but also influences the function of other critical receptors, playing a pivotal role in B-cell-mediated immune regulation and memory formation.

Dendritic cells

DCs are crucial antigen-presenting cells that play a central role in initiating adaptive immune responses, inducing immune tolerance and maintaining immune homeostasis. They orchestrate immune surveillance by capturing, processing and presenting antigens to T-cells. Palmitoylation, as a reversible post-translational lipid modification, has emerged as an important regulator of DC function, migration and immune activation. In DCs, palmitoylation of pattern recognition receptors (e.g. TLRs) and co-stimulatory molecules contributes to antigen uptake and DC activation, especially during viral or bacterial infections [44]. In DCs, zDHHC18 negatively regulates cyclic GMP-AMP synthase (cGAS) activity by catalysing palmitoylation at Cys474, thereby limiting type I interferon (IFN) production in response to cytosolic DNA [45].

Palmitoylation and ARDS

Emerging evidence indicates that palmitoylation has garnered increasing attention as a crucial lipid post-translational modification involved in cellular functions, immune responses, signal transduction and lung tissue injury. Palmitoylation regulation affects immune cell function and plays a pivotal role in cell migration, inflammatory responses and tissue repair processes during lung injury (figure 2). ARDS is clinically heterogeneous and manifests in two main subtypes: hyperinflammatory and hypoinflammatory. Different pathophysiological states resulting from selective activation of palmitoylation signalling may be the central driving mechanism for the differentiation of the two.

FIGURE 2.

Multi-pathway roles and therapeutic targets of protein palmitoylation in acute respiratory distress syndrome (ARDS) pathogenesis. Zinc finger aspartate-histidine-histidine-cysteine motif-containing (zDHHC)-mediated palmitoylation regulates inflammasome activation, pyroptosis, immune checkpoint expression, fibroblast activation, endothelial barrier stability and cellular metabolism, offering diverse therapeutic targets. 2-BP: 2-bromopalmitate; Cys: cysteine; GSDMD: gasdermin D; IL-1β: interleukin 1β; LPS: lipopolysaccharide; MYD88: myeloid differentiation primary response 88; NF-κB: nuclear factor κB; NLRP3: nucleotide-binding oligomerisation domain, leucine-rich repeat, and pyrin domain-containing 3; PD-L1: programmed death ligand 1; PECAM-1: platelet endothelial cell adhesion molecule 1; PKM2: pyruvate kinase M2 isoform; ROS: reactive oxygen species; TLR4: Toll-like receptor 4.

Inflammasome activation and cytokine storm

The inflammatory response plays a dual role in host defence. A moderate inflammatory response is essential for pathogen clearance, infection control and tissue repair, serving as a crucial innate immune mechanism. However, an excessive inflammatory response can lead to tissue damage, sepsis and multi-organ failure, significantly increasing the risk of mortality. In the pathogenesis of ALI and ARDS, uncontrolled local or systemic inflammation plays a central role. Upon stimulation, immune cells release large quantities of pro-inflammatory cytokines such as IL-1, IL-6 and tumour necrosis factor α (TNF-α), which interact to recruit and infiltrate additional inflammatory cells into lung tissue. This cascade activates intracellular signalling pathways, further amplifying the inflammatory response and creating a vicious cycle that ultimately results in a cytokine storm [46]. In the inflammatory process of ALI/ARDS, multiple signalling pathways are involved, including NF-κB, MAPK, TLRs and JAK/STAT [47–49]. Immune cells such as neutrophils, macrophages and T-cells play critical roles in the pathological progression of ALI. Palmitoylation modulates immune cell function by altering surface receptor expression and intracellular signalling pathways. For instance, in lipopolysaccharide (LPS)-induced lung injury models, palmitoylation has been shown to inhibit excessive neutrophil activation and migration, thereby reducing alveolar–capillary barrier damage [12].

Nucleotide-binding oligomerisation domain-containing 1 (NOD1) and 2 (NOD2) play a key role in inflammatory responses by activating immune cells and promoting inflammasome formation [50]. Upon recognising pathogen-associated molecular patterns, such as bacterial DNA, or endogenous damage-associated molecular patterns, NOD1 and NOD2 interact with receptor-interacting protein 2 (RIP2), triggering NF-κB and MAPK signalling pathways. This leads to the release of pro-inflammatory cytokines, including TNF-α and IL-6, promoting immune cell activation and migration, thereby exacerbating both local and systemic inflammation [51]. zDHHC5-mediated palmitoylation of NOD1 and NOD2 is essential for their proper recruitment to bacterial entry sites and phagosomes, which is crucial for optimal bacterial peptidoglycan sensing and subsequent activation of NF-κB signalling [52].

cGAS is a cytosolic DNA sensor that mediates signal transduction through the stimulator of interferon gene (STING)/TANK-binding kinase 1/interferon regulatory factor 3 (IRF3) axis, leading to the production of type I IFNs and pro-inflammatory cytokines [53, 54]. In lung injury, cGAS detects intracellular DNA damage or pathogenic DNA, thereby activating the downstream STING signalling pathway. This process promotes the production of IFNs and inflammatory cytokines by immune cells, further activating the NLRP3 inflammasome and initiating inflammatory responses [55, 56]. Shi et al. [45] demonstrated that zDHHC18-deficient mice exhibit resistance to DNA virus infection. Palmitoylation of cGAS is negatively regulated by zDHHC18, thereby suppressing cGAS activation, which is critical for controlling excessive inflammatory responses. Recent studies have explored targeting the palmitoylation site of STING as a potential therapeutic approach. STING palmitoylation inhibitors have been shown to disrupt the assembly of polymerised STING complexes in the Golgi apparatus, thereby suppressing downstream inflammatory signalling and reducing type I IFN release, highlighting their potential as therapeutic agents for STING-dependent inflammatory diseases [57, 58]. Additionally, Liu et al. [59] used zDHHC11 knockout mouse models to validate the role of zDHHC11 in STING-mediated innate immune responses against DNA viruses. Their findings suggest that zDHHC11 enhances the interaction between MITA (also known as STING) and IRF3, playing a crucial role in antiviral immune defence.

NLRP3 is a key pattern recognition receptor that triggers inflammasome formation, leading to caspase-1 activation and the secretion of IL-1β and IL-18 [60, 61]. However, the mechanisms preventing its sustained activation remain unclear. zDHHC5 and zDHHC12 mediate the palmitoylation of NLRP3 at different cysteine residues. These modifications occur at distinct times and locations. zDHHC5 catalyses palmitoylation at Cys130, enhancing NLRP3 oligomerisation and interaction with NEK7. NEK7 is a kinase essential for inflammasome assembly. This process promotes ASC speck formation, caspase-1 activation, and IL-1β maturation and secretion [28]. In contrast, zDHHC12 mediates palmitoylation. This modification recruits HSC70, triggering chaperone-mediated autophagy and subsequent lysosomal degradation of NLRP3. This negative feedback loop prevents prolonged inflammation [62]. Such dual regulatory mechanisms ensure precise modulation of NLRP3 inflammasome activity, effectively balancing pathogen clearance with the prevention of excessive tissue damage [63].

Recent studies suggest that differences in palmitoylation activity may underlie the pathophysiological divergence between ARDS sub-phenotypes. Calfee et al. [64] proposed two clinically relevant phenotypes, hyperinflammatory and hypoinflammatory, distinguished by systemic inflammation, organ dysfunction and prognosis. In hyperinflammatory ARDS, pro-inflammatory palmitoylation appears to be more active. zDHHC6-mediated palmitoylation of MyD88 in neutrophils enhances TLR signalling and promotes the release of cytokines such as IL-6 and TNF-α, while zDHHC5 catalyses NLRP3 palmitoylation at Cys130, promoting inflammasome assembly and IL-1β maturation. Palmitoylation of gasdermin D (GSDMD) by zDHHC5/9 facilitates membrane translocation and pore formation, further contributing to pyroptotic cell death. These processes may synergistically drive cytokine storm and tissue damage [65]. By contrast, the hypoinflammatory subtype may reflect enhanced regulatory palmitoylation. zDHHC12 modifies NLRP3, recruiting HSC70 and promoting lysosomal degradation, thereby limiting inflammasome activation. In Tregs, palmitoylation of Foxp3 by DHHC2 enhances its transcriptional activity and supports immune suppression. Palmitoylation-mediated modulation of Wnt ligands may also promote epithelial repair in the late phase of ARDS.

These observations suggest that palmitoylation signatures may differ between ARDS subtypes, and future interventions may need to account for both inflammatory profile and disease stage when targeting palmitoylation pathways.

Oxidative stress

Palmitoylation also influences lung injury by regulating oxidative stress and apoptosis. Oxidative stress plays a crucial role in the pathogenesis and progression of ARDS [66]. By modulating the antioxidant capacity of macrophages, palmitoylation may reduce oxidative damage, thereby mitigating lung injury.

Pyroptosis, a lytic form of programmed cell death driven by inflammatory signals, plays dual roles in innate immunity and pathological inflammation. Caspase-4/11 is activated by LPS through noncanonical inflammasome pathways, cleaving GSDMD into an N-terminal pore-forming domain (GSDMD-NT) and a C-terminal autoinhibitory fragment [66]. Mitochondrial reactive oxygen species oxidise palmitoyltransferases zDHHC5 and zDHHC9, markedly enhancing their enzymatic activity. Activated zDHHC5/9 catalyse palmitoylation of GSDMD-NT at Cys191 (human) or Cys192 (mouse), enabling its translocation to cardiolipin-enriched membrane domains [67]. Palmitoylated GSDMD-NT oligomerises into 10–20 nm pores, triggering osmotic cell lysis and IL-1β release [68]. Small-molecule inhibitors targeting zDHHC5 selectively block GSDMD pore formation without perturbing upstream inflammasome signalling, offering a precision strategy to attenuate pyroptosis-driven pathology in ARDS.

Immune regulation

Palmitic acid (PA) alleviates lung injury by modulating immune response balance. Under inflammatory conditions, PA regulates the palmitoylation of immune receptors such as TLRs and NLRs, thereby influencing their signalling functions and preventing excessive inflammatory responses. Through this mechanism, palmitoylation promotes immune tolerance, reduces excessive cytokine release and mitigates pulmonary inflammation.

In lung injury, immune cells recognise pathogens or damage-associated signals through Fc receptors and pattern recognition receptors, such as TLRs, thereby initiating immune responses [65, 69]. The TCR-associated protein (TRAP) family consists of key signalling molecules, and a subset known as palmitoylated TRAPs (pTRAPs) plays additional roles in innate immune cells. pTRAPs target lipid rafts, annexin-rich microdomains and membrane protein clusters, acting as scaffolds to recruit signalling kinases, particularly Src and Syk family members, as well as Csk and other effectors, to facilitate spatiotemporal regulation of immune signalling [70, 71]. Through this mechanism, pTRAPs modulate signal transduction and influence downstream immune responses, including the selective release of inflammatory cytokines and other mediators. Recent studies indicate that in addition to interacting with various transmembrane partners, pTRAPs can directly bind to innate immune receptors, further enhancing their role in immune regulation. Therefore, pTRAPs serve as critical multifunctional scaffolds in fundamental pathways of innate immune responses. They facilitate the localisation of these receptors within lipid rafts, enabling interactions with signalling kinases such as Src and Syk family members, as well as other effectors like Csk, thereby promoting immune response activation [72].

Fas is a transmembrane receptor and its ligand, FasL, is primarily expressed on the surface of certain immune cells, including T-cells, natural killer cells and DCs. Upon binding to Fas, FasL recruits and interacts with Fas-associated death domain protein, which subsequently activates caspase-8, a cysteine protease responsible for initiating apoptosis. Once activated, caspase-8 triggers the downstream caspase cascade, including caspase-3, leading to the cleavage of multiple intracellular substrates, nuclear fragmentation and, ultimately, cell death [73]. In lung injury, FasL-mediated cell death is critical for maintaining immune homeostasis and eliminating virus-infected or transformed cells. Due to its potent cytotoxic effects, the surface expression of FasL is tightly regulated to prevent excessive immune activation and tissue damage [74, 75]. Studies have shown that a disintegrin and metalloprotease 10 (ADAM10)-mediated processing of FasL can inhibit FasL-induced cell death, and this process is precisely regulated by FasL membrane localisation, interactions and modifications. Research indicates that FasL processing preferentially occurs within cholesterol- and sphingolipid-enriched nanodomains (lipid rafts), where FasL effectively interacts with the Fas receptor. Efficient FasL processing depends on the interaction between Fas and FasL within these microdomains. Furthermore, FasL palmitoylation occurs within its transmembrane domain, a modification that is essential for both its cytotoxic activity and proper processing [76]. This pathway regulates immune cell apoptosis, preventing excessive immune activation. Cruz et al. [55] demonstrated that palmitoylation of membrane-proximal cysteine residues allows Fas to localise to lipid raft microdomains and induce apoptosis in cell lines. In contrast, the Fas C194V mutant (a depalmitoylated Fas variant) failed to effectively induce apoptosis in primary mouse T-cells, B-cells and DCs. However, despite its inability to trigger apoptosis efficiently, this mutant was still capable of promoting initial T-cell differentiation. Although the Fas C194V mutant could not effectively induce cell death, it prevented lymphoproliferation and autoimmunity in Fas-deficient mice. These findings suggest that Fas palmitoylation is crucial for apoptosis induction in primary immune cells. Furthermore, Fas may prevent autoimmunity through alternative mechanisms, such as regulating immune cell differentiation and immune tolerance.

Endothelial system protection

Palmitoylation contributes to lung protection by modulating immune responses, reducing oxidative stress, and enhancing pulmonary vascular barrier function, thereby alleviating lung injury and promoting airway repair. Studies have shown that palmitoylation modifications in pulmonary vascular endothelial cells regulate endothelial barrier integrity. Specifically, modulator of immune recognition 2 (MIR2) function is inhibited in the presence of 2-BP, a palmitoylation inhibitor. Biochemical analysis revealed that MIR2 undergoes direct palmitoylation at Cys146, which is essential for its regulatory function. Blocking MIR2 disrupts its ability to downregulate major histocompatibility complex class I and platelet endothelial cell adhesion molecule 1, a key molecule involved in vascular endothelial function and immune cell migration [77, 78]. Palmitoylation enhances immune receptor localisation within lipid rafts, improving their responsiveness to immune cells and pathogens. In lung injury, lipid rafts serve as critical microdomains in endothelial cells, playing a key role in regulating lung injury repair [79]. Beard et al. [80] demonstrated that inhibiting palmitoyltransferases, particularly zDHHC21, alleviates endothelial barrier dysfunction and reduces leukocyte adhesion, thereby mitigating lung injury and improving pulmonary pathology. Further research has shown that palmitoylation of phospholipase C β1 is a key process in endothelial inflammation, because its activity and palmitoylation status directly influence endothelial function and inflammatory responses. In endothelial cells, treatment with the palmitoyltransferase inhibitor 2-BP prevents PA-induced endothelial damage, whereas ML349, a depalmitoylase inhibitor that promotes palmitoylation, exacerbates PA-induced injury. Proteomic analysis of palmitoylated proteins has identified PKM2 as a key palmitoylation target involved in PA-induced endothelial damage, with Cys31 being the primary palmitoylation site. Notably, the PKM2-C31S mutant (substituting cysteine with serine) prevents PA-induced endothelial injury, further confirming the role of palmitoylation in endothelial dysfunction. Endothelial-specific adeno-associated virus carrying the C31S mutant of pyruvate kinase M2 targeted to endothelial cells (AAV-C31S PKM2^endo) gene therapy has been shown to improve cardiovascular dysfunction in PA-induced ApoE^−/− mice. Mechanistically, PKM2 palmitoylation at Cys31 impairs its enzymatic activity and disrupts glycolytic flux in endothelial cells, contributing to barrier dysfunction in pulmonary injury. Ultimately, zDHHC13 was identified as the palmitoyltransferase responsible for PKM2 palmitoylation. Collectively, these findings suggest that Cys31 palmitoylation of PKM2 plays a critical role in PA-induced endothelial dysfunction [23].

The therapeutic potential of palmitoylation for ARDS

Palmitoylation is a crucial post-translational modification that plays a key role in inflammation regulation, oxidative stress and cellular repair. Emerging evidence indicates a link between aberrant palmitoylation and the pathogenesis and progression of lung injuries, including ARDS and pulmonary fibrosis, leading to the identification of potential therapeutic strategies targeting this modification.

Regulation of inflammasomes and pyroptosis

The NLRP3 inflammasome is a major driver of ALI, and its activation depends on palmitoylation at specific cysteine residues. Studies have shown that zDHHC5 and zDHHC7 catalyse NLRP3 palmitoylation, promoting inflammasome assembly and IL-1β release [63]. The development of small-molecule inhibitors targeting palmitoylation has shown promise in preclinical models. 2-BP inhibits zDHHC enzymes, thereby reducing GSDMD palmitoylation at Cys191 and preventing its membrane localisation [81]. Additionally, NLRP3 inhibitors such as MCC950 and dapansutrile are currently under clinical investigation for their potential to suppress excessive pulmonary inflammation, further highlighting inflammasome-targeted strategies as a promising approach for lung injury treatment.

PD-L1 regulation and immunotherapy

Palmitoylation of PD-L1 enhances its membrane stability, and inhibition of zDHHC3, a key enzyme mediating PD-L1 palmitoylation, has been found to accelerate PD-L1 degradation, with palmitoylated PD-L1 recruiting SH2 domain-containing protein tyrosine phosphatase-2, which inhibits T-cell function and thereby enhances immune clearance of tumour cells and pathogen-infected cells [37]. The marine-derived DHHC3 inhibitor benzosceptrin C disrupts palmitoylation of PD-L1 and acts synergistically with anti-PD-1 therapies in preclinical models, and this compound is currently undergoing preclinical evaluation, showing great potential for immunotherapy applications [82].

Fibrosis regulation and lung repair

The Wnt signalling pathway plays a crucial role in fibroblast activation during lung repair, and the secretion and activity of Wnt ligands depend on Porcupine-catalysed palmitoylation. Porcupine inhibitors, such as RXC006/AZD5055, effectively block Wnt signalling, thus reducing fibroblast proliferation and collagen deposition and consequently attenuating pulmonary fibrosis. This therapeutic strategy has advanced into clinical trials for idiopathic pulmonary fibrosis [83]. Additionally, the broad-spectrum palmitoylation inhibitor 2-BP has demonstrated antifibrotic effects in preclinical animal models, highlighting its therapeutic potential.

Building upon these insights, combination regimens may enhance therapeutic efficacy. For instance, Porcupine inhibitors could be paired with established antifibrotic agents such as pirfenidone or nintedanib to achieve synergistic suppression of fibroblast activation and extracellular matrix remodelling. Moreover, in patients exhibiting overlapping inflammatory and fibrotic subtypes, a sequential treatment strategy may be considered, initially targeting inflammasome activation during the acute phase (e.g. via zDHHC5/9 inhibitors) followed by Wnt blockade during the repair phase. To improve pulmonary targeting and minimise systemic exposure, inhalable nanoparticle-based delivery systems may be employed. These formulations can enhance alveolar retention, particularly within macrophages and fibroblasts, thereby maximising on-site pharmacological activity while limiting off-target toxicity.

In the Ras/MAPK pathway, palmitoylation governs the spatial distribution of H-Ras and N-Ras between raft and non-raft domains, thereby influencing downstream Raf isoform preference and MAPK activation. Misregulation of this cycle disrupts signalling fidelity and may exacerbate injury responses.

Temporal dynamics of palmitoylation in ARDS

Palmitoylation is a dynamic and reversible lipid modification, and its regulatory roles in ARDS may vary significantly over the course of disease progression. Given the temporal heterogeneity of ARDS, ranging from acute inflammation to resolution and potential fibrosis, it is essential to consider the time-dependent shifts in palmitoylation activity and their functional implications. In the early phase of ARDS, typically within the first 72 h following injury, pro-inflammatory palmitoylation events tend to dominate. These include palmitoylation of innate immune signalling molecules and pyroptosis-related proteins, which collectively amplify cytokine production, inflammasome activation and tissue injury. This stage may benefit from targeted inhibition of specific palmitoyltransferases involved in immune overactivation. As the disease progresses into the resolution or fibrotic phase, the landscape of palmitoylation shifts. Enzymatic pathways that suppress excessive inflammation, such as those mediating degradation of inflammatory sensors or promoting Treg function, become more prominent. In parallel, palmitoylation also contributes to tissue repair and fibroblast activation, particularly through pathways regulating Wnt signalling. At this stage, broad inhibition of palmitoylation may interfere with beneficial reparative responses and exacerbate fibrotic remodelling.

These temporal dynamics highlight the need for stage-specific therapeutic strategies targeting palmitoylation. Early-phase interventions may focus on blocking pro-inflammatory palmitoylation nodes to mitigate ALI, while later phases may require selective modulation, or even enhancement, of palmitoylation pathways that support resolution and repair. Future studies should aim to delineate these time-dependent patterns in patient samples and animal models to inform the design of precision-timed interventions.

Conclusions and perspectives

ARDS remains a life-threatening condition characterised by uncontrolled inflammation, oxidative stress and disrupted tissue repair, with limited therapeutic options and high mortality rates. Over the past decade, the dynamic and reversible nature of palmitoylation, a lipid post-translational modification, has emerged as a critical regulator of ARDS pathogenesis. This modification, mediated by zDHHC, orchestrates immune cell activation, inflammasome assembly, endothelial barrier integrity and fibrotic remodelling. However, the dual roles of palmitoylation in both promoting host defence and exacerbating pathological injury underscore the complexity of targeting this pathway for therapeutic intervention. Preclinical studies have identified promising modulatory strategies, but the translational bottlenecks, especially biomarker availability, drug delivery precision and subtype differentiation, remain significant [84]. Addressing these challenges will require interdisciplinary approaches integrating advanced technologies, rigorous clinical validation and innovative drug design.

The regulation of immune responses by palmitoylation is a cornerstone of ARDS pathophysiology. Neutrophils, macrophages, T-cells and B-cells rely on palmitoylation to fine-tune their activation thresholds and functional outputs. For example, zDHHC3-mediated MyD88 palmitoylation amplifies TLR signalling in neutrophils, driving cytokine storms that exacerbate alveolar damage. Conversely, palmitoylation of TLR4 in macrophages promotes M1 polarisation and cytokine production; compensatory IL-10 expression may occur in prolonged inflammatory states. Similarly, TCR signalling and PD-L1 stability are modulated by palmitoylation, with implications for adaptive immunity and immune tolerance. These findings suggest that selective inhibition or enhancement of specific zDHHC enzymes could rebalance immune responses in ARDS. Nevertheless, the lack of cell type-specific inhibitors and the ubiquitous expression of zDHHCs pose challenges for therapeutic precision. For instance, 2-BP, a broad-spectrum palmitoyltransferase inhibitor, suppresses neutrophil infiltration but may inadvertently disrupt neuronal or metabolic pathways. Future efforts must prioritise the development of enzyme-selective inhibitors, guided by structural insights into zDHHC catalytic domains and substrate binding pockets. Recent advances in cryo-electron microscopy and molecular dynamics simulations could accelerate this process, enabling rational drug design to target pathogenic zDHHC isoforms while sparing their physiological counterparts.

Inflammasome activation and pyroptosis represent another critical axis regulated by palmitoylation in ARDS. The NLRP3 inflammasome, a key driver of lung injury, is dynamically controlled by zDHHC5 and zDHHC12 through opposing mechanisms. While zDHHC5-mediated palmitoylation at Cys130 facilitates NLRP3 oligomerisation and inflammasome assembly, zDHHC12 promotes NLRP3 degradation via chaperone-mediated autophagy. This dual regulation ensures a balanced inflammatory response, but dysregulation can lead to uncontrolled cytokine release or immunosuppression. Similarly, GSDMD palmitoylation by zDHHC5/9 enables pore formation and pyroptosis, a lytic cell death process that amplifies lung injury. Inhibitors like ML349, which block GSDMD membrane translocation without affecting upstream signalling, exemplify the potential for precision therapeutics. However, the interplay between palmitoylation and other post-translational modifications, such as ubiquitination or phosphorylation, remains poorly understood. Multi-omics approaches combining palmitoylome profiling with phosphoproteomics and ubiquitinomics could unravel these complex regulatory networks, identifying nodes for synergistic intervention. Furthermore, the role of palmitoylation in mitochondrial function and oxidative stress warrants deeper exploration. For example, palmitoylation of PKM2 in endothelial cells impairs glycolysis and exacerbates barrier dysfunction, whereas zDHHC13 knockdown rescues these defects. Targeting such pathways could mitigate oxidative damage while preserving immune function, but the tissue-specific effects of these interventions must be carefully evaluated.

A major translational hurdle lies in the absence of reliable biomarkers for patient stratification and response monitoring. Direct assessment of protein palmitoylation in clinical samples remains limited due to the need for invasive sampling and unstable lipid linkages. Emerging technologies, including microfluidic mass spectrometry and acyl-probe systems, now allow trace detection in serum or bronchoalveolar lavage fluid with increased feasibility. Meanwhile, indirect indicators, such as zDHHC5/zDHHC12 expression or downstream inflammatory mediators (e.g. IL-6, NLRP3), may reflect palmitoylation dynamics in situ. Preliminary data from metabolic ARDS suggest that serum PKM2 palmitoylation may correlate with mortality, while soluble PD-L1 and zDHHC3 co-expression patterns, borrowed from oncology studies, may help tailor immunotherapies. Nonetheless, these biomarkers remain exploratory, and large, multi-cohort validations are urgently needed. Moving forward, an integrated biomarker framework that spans upstream zDHHC expression, downstream effectors (e.g. inflammasome mediators) and direct palmitoylation profiles will be crucial for defining actionable sub-phenotypes and monitoring therapy. In parallel, combination approaches targeting both immunoinflammatory and structural arms of ARDS are emerging. For example, zDHHC3 inhibition, proposed to destabilise PD-L1, may hypothetically enhance anti-PD-1 efficacy in infection-associated ARDS, although this strategy is currently extrapolated from oncology research and lacks ARDS-specific validation. Similarly, Porcupine inhibitors (e.g. RXC006) have shown antifibrotic effects in preclinical pulmonary fibrosis models, but their utility in ARDS-associated remodelling requires further investigation. Nevertheless, potential risks remain; chronic suppression of palmitoylation has been linked to altered synaptic signalling and metabolic imbalance in neurological contexts, underscoring the need for careful toxicity evaluation in ARDS applications.

Emerging technologies offer unprecedented opportunities to overcome these challenges. Single-cell RNA sequencing and spatial transcriptomics can map zDHHC expression patterns across lung cell populations, revealing understudied roles in alveolar epithelial cells or fibroblasts. CRISPR–Cas9 screens in ARDS animal models may identify novel zDHHC/depalmitoylase pairs that drive disease progression. Meanwhile, organoid-based systems could model patient-specific responses to palmitoylation modulators, bridging the gap between preclinical and clinical research. The integration of artificial intelligence into drug discovery pipelines could further accelerate the identification of selective inhibitors. Machine-learning algorithms trained on structural data from zDHHC enzymes might predict inhibitor binding affinities or off-target risks, streamlining the development of safer therapeutics.

In conclusion, palmitoylation represents a context-dependent and stage-specific modulator in ARDS. Its dual capacity to support immune resolution or drive pathological inflammation demands precise temporal and molecular targeting. Future success will depend on deeper mechanistic understanding, biomarker-guided stratification and integration of precision pharmacology to translate palmitoylation from molecular insights to viable clinical targets. Although significant challenges remain, the convergence of technological innovation and mechanistic research has made palmitoylation a transformative frontier for ARDS research. Collaborative efforts among basic scientists, clinicians and bioengineers will be essential to translate this potential into therapies that improve outcomes for ARDS patients worldwide. In summary, palmitoylation is a dynamic, reversible and context-sensitive post-translational modification embedded within complex signalling and regulatory networks. Its functional output is not determined solely by the presence or absence of lipid moieties but by the interplay of enzymatic specificity, substrate configuration, post-translation modification crosstalk and subcellular microenvironments. Future research and therapeutic translation must take this mechanistic intricacy into account.