Introduction

The innate immune response constitutes the initial barrier of host defense against pathogenic bacterial infections. Pattern recognition receptors (PRRs) within host cells can recognize the pathogen-associated molecular patterns (PAMPs) of bacteria and swiftly initiate countermeasures, effectively eliminating intracellular pathogens via phagocytosis and inflammatory responses. The inflammasome activation is a pivotal innate immune defense mechanism in combating bacterial infections.¹ Inflammasomes are macromolecular multimeric protein complexes that function as an activation platform for cysteinyl aspartate specific proteinase (Caspase).² Inflammasomes are adept at recognizing various danger signals within host cells, including PAMPs, microbial-associated molecular patterns (MAMPs), and damage-associated molecular patterns (DAMPs). Upon activation, inflammasomes can trigger pro-inflammatory pathways and antimicrobial responses, leading to the secretion of various pro-inflammatory cytokines and chemokines, as well as the up-regulation of immunoreceptors and cell adhesion molecules.³ According to different activation pathways, inflammasomes can be divided into two categories: canonical and non-canonical inflammasomes.

Canonical inflammasomes are formed by the sensor PRRs, the adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD [caspase activation and recruitment domain]), and the effector pro-Caspase-1.⁴ The activation of canonical inflammasome triggers the self-cleavage and activation of Caspase-1, which subsequently cleaves the pro-inflammatory cytokines pro-IL-1β and pro-IL-18 into their active, secreted forms.⁵ Additionally, the pro-Gasdermin D (pro-GSDMD) is identified as a substrate for Caspase-1/4/5/11, the N-terminal product of GSDMD cleavage can form pores in the plasma membrane. This pore-forming activity leads to a form of pro-inflammatory programmed necrosis known as pyroptosis, which further contributes to the host immune defense by eliminating infected cells.⁶ Among the canonical inflammasomes, Nucleotide-binding oligomerization domain (NOD)-like receptor family pyrin domain-containing protein 1 (NLRP1), NLRP3, and NOD-like receptor family CARD domain containing protein 4 (NLRC4) inflammasomes play pivotal roles as surveillance machinery during bacterial infection.^7–9 They act as front-line sentinels, constantly monitoring for the presence of bacteria and initiating appropriate immune responses. Unlike the canonical inflammasomes, the activation of non-canonical inflammasomes does not rely on PRRs sensors. This divergence in activation mechanisms reflects the complexity and flexibility of the immune system to adapt to different types of threats. Instead, Caspase-4/5 (in humans) and Caspase-11 (in mice) take on the role of pathogen sensors. They recognize and directly bind cytosolic lipopolysaccharide (LPS) through their CARD domains.¹⁰ This direct interaction with LPS bypasses the need for PRR-mediated sensing, allowing for a more rapid response to the presence of intracellular Gram-negative bacteria. Once Caspase-4/5/11 bind to LPS, they then initiate IL-1β proteolytic maturation and pyroptosis in a GSDMD-dependent manner. This process is crucial as it provides a crucial host defense against intracellular Gram-negative bacteria, complementing the functions of canonical inflammasomes in maintaining immune homeostasis.¹¹

In addition to its critical role in bacterial defense, the NLRP3 inflammasome has emerged as a central mediator in multiple human pathologies, underscoring its potential as a therapeutic target. Recent studies highlight its involvement in vascular diseases, where excessive NLRP3 activation drives atherosclerotic lesion formation, vascular dysfunction, and aneurysm development through Caspase-1- dependent IL-1β/IL-18 secretion and pyroptosis.¹² Inflammatory bowel diseases such as Crohn’s disease (CD) are also linked to NLRP3 inflammasome hyperactivation and distinct blood microbiome signatures, including enrichment of Staphylococcus sciuri and altered IL-1β profiles.¹³ These findings highlight NLRP3 as a potential diagnostic biomarker and suggest that microbial-targeted interventions may modulate inflammatory responses in CD. Emerging evidence further implicates NLRP3 in neurodegenerative disorders, with studies demonstrating its contribution to amyloid-β aggregation and neuroinflammation in Alzheimer’s disease.¹⁴ Moreover, metabolic dysfunction-associated steatotic liver disease (MASLD) exhibits a bidirectional relationship with NLRP3, where gut microbiota-derived metabolites promote inflammasome activation in hepatocytes, exacerbating hepatic steatosis and fibrosis.¹⁵ These findings collectively position the NLRP3 inflammasome as a key node in inflammation-driven pathologies, prompting ongoing development of targeted therapies including small molecule inhibitors and microbiome modulation strategies.

Salmonella enterica is a flagellated facultative anaerobic Gram-negative bacterium, more than 2600 serovars have been characterized based on the differences in flagellin (H) and lipopolysaccharide (O) antigens.¹⁶ Salmonella is one of the most important and dangerous foodborne zoonotic pathogens globally, accountable for approximately 93.8 million foodborne disease cases and roughly 155,000 fatalities annually,¹⁷ the contaminated poultry, eggs, vegetables, and dairy products are the most common sources of infection.¹⁸ After invading the host through the gastrointestinal tract, Salmonella crosses the epithelial barrier by invading intestinal epithelial cells or microfold cells and subsequently disseminates to systemic sites by invading nearby phagocytes.¹⁹ The ability of Salmonella to invade host cells is dependent on the Salmonella pathogenicity island-1 (SPI-1)-encoded type III secretion system-1 (T3SS-1).²⁰ Once internalized, Salmonella swiftly establishes a specialized Salmonella-containing vacuole (SCV) via SPI-2-encoded T3SS-2 for replication.^21,22 Over 60 effector proteins are secreted via T3SS, aiding in maintaining SCV homeostasis, modulating host cells progression, and evading host immune responses to achieve persistent infection.²³

Previous studies have demonstrated that the canonical NLRP3 and NLRC4 inflammasome, as well as the non-canonical Caspase-4/5/11 inflammasome are mainly activated during Salmonella infection.²⁴ The activation of these inflammasomes plays a crucial role in limiting Salmonella infection. Mice lacking Caspase-1/11 succumb to Salmonella infection more rapidly than wild-type (WT) mice, exhibiting significantly elevated bacterial burdens in organs.^25,26 Given the antimicrobial properties of inflammasomes, Salmonella has also developed various mechanisms to avoid or delay the activation of inflammasome.

Currently, the canonical NLRC4 and non-canonical Caspase-11 inflammasome pathways have been established as playing a crucial role in resisting Salmonella infection of the gut through inflammasome-dependent sloughing of infected epithelial cells.^10,27–29 However, several unresolved questions persist regarding NLRP3 inflammasome activation during Salmonella infection. Foremost among these is the divergent role of NLRP3 in different infection models, with conflicting evidence regarding its necessity for host defense. In systemic Salmonella infection models, NLRC4 and NLRP3 have been shown to exhibit redundant functions in inducing Caspase-1 activation, with mice deficient in both NLRC4 and NLRP3 displayed significantly increased susceptibility to infection.³⁰ Conversely, another study suggests that NLRP3 is dispensable during Salmonella infection, as there are no significant differences in bacterial counts, cytokine levels, and histological inflammation between WT and Nlrp3^−/− mice.³¹ In contrast, numerous studies have emphasized the indispensable role of NLRP3 inflammasome activation in host defense against Salmonella infection.^32–34 Moreover, the molecular mechanisms underlying NLRP3 recognition of Salmonella remain incompletely characterized, particularly the specific ligands or upstream signaling events that trigger its activation. The interplay among canonical, non-canonical, and alternative NLRP3 pathways during infection also requires further clarification. Equally critical are the bacterial evasion strategies targeting NLRP3, with many effector proteins and metabolic adaptations yet to be fully understood. Addressing these gaps will advance our understanding of host-pathogen interactions and inform therapeutic development. In this review, we delve into recent findings concerning the role of NLRP3 inflammasome in resisting Salmonella infection and explore the complex interplay of detection and evasion between NLRP3 inflammasome and Salmonella.

Mechanisms of NLRP3 Inflammasome Activation

Canonical NLRP3 Inflammasome

NLRP3 is one of the most extensively studied NLR family proteins, which consists of an amino-terminal pyrin domain (PYD), a central nucleotide-binding and oligomerization domain (NACHT), and a C-terminal leucine-rich repeat (LRR) domain.³⁵ Previous studies suggest that the LRR domain mediates auto-inhibition of NLRP3 by folding back into the NACHT domain.³⁶ However, a recent study indicates that the LRR domain is dispensable for NLRP3 auto-inhibition.³⁷ Notably, NIMA-related kinase (NEK7), a serine/threonine kinase contributing to mitosis, has been found to play an essential role in NLRP3 inflammasome activation through direct binding to the LRR domain of NLRP3.³⁸ The activation of the canonical NLRP3 inflammasome requires a priming step and an activation step. First, upon recognition of PAMPs or DAMPs by PRRs, transcription factors such as NF-κB are activated, leading to the up-regulation of NLRP3 and pro-IL-1β expression.^39,40 Importantly, multiple post-translational modifications of NLRP3, including ubiquitination, phosphorylation, sumoylation, and S-nitrosylation, also prime NLRP3 for activation while maintaining its auto-inhibited state.^41,42 Second, the activation step involves the recognition of NLRP3 agonists, leading to the assembly and activation of NLRP3 inflammasome. Unlike PRRs, which typically recognize one or several structurally similar stimuli, NLRP3 can be activated by a diverse range of structurally and chemically dissimilar agonists, including PAMPs such as bacterial, fungal, and viral toxins, DAMPs such as extracellular ATP, particulates such as silica and uric acid crystals.^43,44 Upon stimulation, NLRP3 oligomerizes through homotypic interactions between NACHT domains. This oligomerized NLRP3 then recruits ASC through homotypic PYD-PYD interactions.³⁹ Following ASC oligomerization and speck formation, ASC recruits pro-Caspase-1 through CARD-CARD interactions, initiating pro-Caspase-1 self-cleavage and activation.⁴⁵ Active Caspase-1 subsequently cleaves and activates GSDMD, which triggers pyroptosis, and converts pro-IL-1β and pro-IL-18 into mature pro-inflammatory cytokines.⁴⁶

It is noteworthy that the precise mechanism by which NLRP3 recognizes its various stimuli remains elusive, as no direct interaction between NLRP3 and these known stimuli has been observed. Despite the similarity in the terminal signaling events triggered by these stimuli, the initial mechanisms are likely to differ. It is suggested that NLRP3 senses common upstream cellular events induced by these stimuli, although the identity of these signal events remains controversial within the field. Proposed upstream cellular events include ionic flux,^47–49 lysosomal rupture,^50,51 reactive oxygen species (ROS) production,^52,53 and mitochondrial dysfunction.^54–56 Although NLRP3 stimuli can activate inflammasome signals via the aforementioned upstream signal events, none of these signal events is applicable to all NLRP3 stimuli. Therefore, the specific mechanisms by which NLRP3 senses these stimuli and how the NLRP3 inflammasome is assembled and activated remain areas requiring further characterization.

Non-Canonical NLRP3 Inflammasome

Cytoplasmic LPS triggers non-canonical Caspase-4/5/11 inflammasome activation, leading to the cleavage of GSDMD and pyroptosis. Notably, Caspase-11 does not participate in the cleavage and maturation of pro-inflammatory cytokines IL-1β and IL-18.⁵⁷ Human Caspase-4 resembles Caspase-1 in its ability to cleave GSDMD and pro-IL-18, but it does not cleave pro-IL-1β. The ability of Caspase-5 to cleave both pro-IL-18 and pro-IL-1β is extremely limited, closely resembling that of Caspase-11.⁵⁸ Activated Caspase-4/5/11 and cleaved GSDMD subsequently trigger the NLRP3-ASC-Caspase-1 signaling pathway, a process known as the non-canonical NLRP3 inflammasome activation, which is crucial for the subsequent maturation and secretion of IL-18 and IL-1β.⁵⁹ This non-canonical NLRP3 inflammasome activation does not necessitate a priming signal and relies on Caspase-11 for functionality. However, the exact mechanism of non-canonical NLRP3 inflammasome activation remains controversial and not fully characterized. Previous studies suggest that Caspase-11 forms a complex with NLRP3/ASC and pro-Caspase-1.^57,60 It is proposed that Caspase-11 and NLRP3 can mutually activate each other through direct interaction between the scaffold domain of Caspase-11 and the LRR/PYD domain of NLRP3, which requires concurrent detection of bacterial mRNA by NLRP3 and binding of LPS by Caspase-11.⁵⁹ This interaction is reported to occur upstream of inflammasome formation and pro-Caspase-11 cleavage. However, other studies have demonstrated that Caspase-11 activation and GSDMD cleavage are upstream signals of NLRP3 activation.^11,61–63 Thus, the exact sequence of events leading to non-canonical NLRP3 inflammasome activation remains an area of ongoing research.

After being cleaved by activated Caspase-4 and Caspase-11, the N terminus of GSDMD forms pores on the cell membrane, resulting in K⁺ efflux, which is crucial for subsequent activation of the non-canonical NLRP3 inflammasome.^63,64 Additionally, Caspase-11 can activate pannexin-1 via catalytic cleavage, thereby promoting ATP efflux, which in turn stimulates NLRP3 inflammasome activation.⁶⁵ Recently, the nuclear orphan receptor Nur77 has been identified as a receptor for cytoplasmic LPS and mitochondrial dsDNA released through Caspase-11-dependent GSDMD pores. When Nur77 simultaneously binds to LPS and mitochondrial dsDNA, it becomes fully activated and directly binds to NLRP3, leading to enhanced NEK7 recruitment, ASC oligomerization, and non-canonical NLRP3 inflammasome activation.⁶² Similar to the canonical NLRP3 inflammasome, there is no singular, unified pathway for the non-canonical NLRP3 inflammasome activation. While some upstream signal events observed in non-canonical NLRP3 inflammasome pathway are consistent with those in the canonical NLRP3 inflammasome pathway, such as K⁺ efflux and ATP efflux.^63,65 The overlap of these upstream signal events between canonical and non-canonical NLRP3 inflammasome activation suggests that the activation of NLRP3 is not triggered by a specific stimulant, the initial signal is an undiscovered minimal common cellular event that is both necessary and sufficient to activate the NLRP3 inflammasome.

Recent studies have revealed that the non-canonical NLRP3 inflammasome can be activated by various lipids beyond LPS.⁶⁶ Specifically, the parasite membrane glycoconjugate lipophosphoglycan (LPG) has been identified as a key molecule in activating Caspase-11 and the non-canonical NLRP3 inflammasome. However, it is noteworthy that additional, yet unidentified molecules are required for this activation, as purified LPG alone fails to activate Caspase-11 or Caspase-4 in vitro.⁶⁷ In environments with limited phosphate availability, bacteria reduce LPS production and synthesize phosphorus-free alternatives, such as ornithine lipid, which has been shown to activate Toll-like receptor 4 (TLR4) and induce K⁺ efflux-dependent non-canonical NLRP3 inflammasome activation.⁶⁸ Interestingly, in the presence of LPS, ornithine lipid can also function as a partial TLR4 antagonist, reducing LPS-induced non-canonical NLRP3 inflammasome activation and cytokine secretion. In dendritic cells (DCs), the oxidized phospholipid 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (oxPAPC) has been found to promote IL-1β secretion through a mechanism dependent on Caspase-11, NLRP3, ASC, and Caspase-1, suggesting that oxPAPC serves as an agonist of the non-canonical NLRP3 inflammasome in DCs.⁶⁹ However, oxPAPC also inhibits LPS-induced pyroptosis and non-canonical NLRP3 inflammasome activation in macrophages by directly binding to Caspase-4 or Caspase-11, thereby competing with LPS binding.⁷⁰ These findings suggest that the activation or inhibition of the non-canonical NLRP3 inflammasome by these lipids is contingent on the cell type, location, and intracellular metabolic environment.

Alternative NLRP3 Inflammasome

Recently, LPS has been discovered to trigger an “alternative” inflammasome in human monocytes, which operates through the NLRP3-ASC-Caspase-1 signaling, distinct from both canonical and non-canonical pathways.⁷¹ This alternative NLRP3 inflammasome activation has been observed in human and porcine monocytes but not in murine cells.⁷² In comparison to the robust activation of canonical and non-canonical NLRP3 inflammasomes, the alternative pathway exhibits weaker activation and lacks classical inflammasome characteristics such as K⁺ efflux dependency, ASC pyroptosome formation, and pyroptosis induction.⁷³ The alternative pathway is triggered by LPS and relies on TLR4, TIR-domain-containing adapter-inducing interferon-β (TRIF), receptor-interacting protein kinase 1 (RIPK1), FAS-associated death domain (FADD), and Caspase-8 upstream of NLRP3 activation.⁵ Notably, the upstream TLR4–TRIF–RIPK1–FADD–Caspase-8 signaling has been shown to play no role in canonical and non-canonical NLRP3 inflammasome activation.⁷¹ However, a recent study indicates that TLR4 and TRIF may not be indispensable upstream components of the alternative pathway, as ligands of TLR1/2, TLR2/6, TLR4 and TLR7 and/or TLR8 all have the potential to trigger the alternative NLRP3 pathway.⁷⁴ Furthermore, the alternative pathway can function independently of RIPK1 kinase activity, suggesting that the mechanism of alternative NLRP3 inflammasome activation requires further characterization.

In addition to LPS, apolipoprotein C3 (ApoC3) has been reported to induce alternative NLRP3 inflammasome activation via forming a heterotrimer with TLR2, TLR4, and SLP adaptor and CSK interacting membrane protein (SCIMP). This heterotrimer triggers Lyn/Syk-dependent calcium entry and ROS formation, leading to the activation of Caspase-8.⁷⁵ A previous study also indicated that ApoC3 activates the NLRP3 inflammasome by enhancing the binding of TXNIP to NLRP3 in the presence of ROS.⁷⁶ Furthermore, a study revealed that heat-killed Salmonella can serve as an alternative NLRP3 inflammasome stimulant in human monocytes. The short isoform of cellular FADD-like IL-1β converting enzyme inhibitory protein (cFLIPs), which is activated by NF-κB, negatively regulates Caspase-8 and halts the alternative NLRP3 inflammasome activation in response to heat-killed Salmonella.⁷⁷ This mechanism effectively prevents cell death caused by otherwise sustained NLRP3 inflammasome activation.

In comparison to the non-canonical and alternative pathways, the canonical pathway of NLRP3 inflammasome activation has been the most extensively researched. The non-canonical NLRP3 inflammasome primarily responds to cytosolic lipids, such as LPS, and is consistently associated with Caspase-4/5/11. Conversely, the alternative NLRP3 inflammasome remains the least studied, involving a multitude of stimuli and Caspases. Therefore, delving deeper into the molecular mechanisms underlying the activation of non-canonical and alternative NLRP3 inflammasomes holds significant promise for identifying the initiating ligands or signals that trigger NLRP3 activation.

NLRP3 Inflammasome Detecting Salmonella

Like other stimuli, the mechanism by which NLRP3 inflammasome recognizes Salmonella remains poorly characterized. It is hypothesized that NLRP3 activation occurs downstream of certain Salmonella-induced signaling events during infection. Salmonella T3SS-1 effector protein SipB has been previously discovered to directly bind and interact with Caspase-1, which is essential for Caspase-1 activation and IL-1β maturation.⁷⁸ A recent study suggests that NLRP3 activation induced by Salmonella is likely due to the activity of the SPI-1 T3SS, as the inflammasome activation level induced by ΔsipB was significantly lower than that of WT Salmonella in Nlrc4^−/− human macrophages.⁷⁹ SipB has also been demonstrated to promote mitochondrial depolarization during Salmonella infection, which is essential for the release of fragmented mitochondrial DNA (mtDNA) and the production of ROS that converts mtDNA to an oxidized form (Ox-mtDNA) (Figure 1). Ox-mtDNA is currently the only identified intermediate product of upstream signal events that serves as the ultimate NLRP3 ligand, or at least a part of it.^55,80 However, another study has reported that mitochondrial damage and mtROS production, which can occur with NLRP3 activators, are not necessary for NLRP3 inflammasome activation.⁴⁷ These findings suggest that the pathway by which NLRP3 inflammasome recognizes Salmonella is not confined to a single specific signal event.

Figure 1 Detection of Salmonella by the NLRP3 inflammasome. Salmonella can be detected by intracellular NLRP3 inflammasome, resulting in pyroptosis and the release of IL-1β and IL-18. Lipopolysaccharide (LPS) stimulates non-canonical NLRP3 inflammasome activation by activating Caspase-4/5/11. The bacterial DNA adenine methylase (Dam) enhances NLRP3 inflammasome activation by promoting the phosphorylation of Jnk. The amyloid curli present in Salmonella biofilms stimulates the TLR2/TLR1 heterocomplex, which causes activation of NF-κB and expression of pro-IL-1β. The endocytosis of curli fibers via internalized TLR2/TLR1 then activates NLRP3 and Caspase-1. The type III secretion system-1 (T3SS-1) effector protein SipB facilitates the generation of mitochondrial reactive oxygen species (mtROS) and the conversion of mitochondrial DNA into its oxidized form (Ox-mtDNA), thereby initiating NLRP3 inflammasome activation. Excessive accumulation of bacterial citrate rapidly activates the NLRP3 inflammasome through an mtROS-dependent pathway. Furthermore, NLRP3 inflammasome recognizes flagellin, bacterial RNA components, and the T6SS-secreted protein ClpV of Salmonella via unknown mechanisms. Figure 1 was independently designed by the authors using Microsoft PowerPoint 2013, with all elements created using native software features.

Indeed, in the context of Salmonella interactions with macrophages, the NLRP3 inflammasome can also respond to Salmonella through SPI-1 and SPI-2 T3SS-independent signals. A recent study has revealed that the Salmonella T6SS core-secreted protein ClpV promotes the expression of NLRP3 through an unknown mechanism (Figure 1). This mechanism is essential for intestinal barrier disruption, bacterial adhesion and colonization.⁸¹ The NLRP3 inflammasome can recognize curli, a bacterial amyloid protein present in Salmonella biofilms, leading to caspase-1 activation and IL-1β production. This response relies on endocytosis of curli fibers via internalized TLR2/TLR1, which activate NF-κB, prime pro-IL-1β transcription and enable NLRP3 activation in macrophages.⁸² The underlying mechanism involves the damage caused in the lysosomal compartment by intracellular amyloid fibers.⁸³ Notably, NLRP3 inflammasome activation in mouse macrophages infected with the curli mutant did not differ significantly from WT-infected macrophages. However, only under curli-inducing growth conditions, the curli mutant displayed significantly reduced IL-1β induction compared to WT Salmonella,⁸² indicating that Salmonella employs mechanisms to limit curli expression during natural infection, thereby evading NLRP3 recognition. Furthermore, the NLRP3 inflammasome can sense Salmonella by recognizing bacterial nucleic acids. Prokaryotic mRNA, a viability-associated PAMP present only in viable bacteria, is responsible for subsequent NLRP3 inflammasome activation, even in the absence of virulence factors.⁸⁴ Further research has shown that the ability of mice and human NLRP3 inflammasome to sense bacterial RNA differs. Human NLRP3 inflammasome can detect all bacterial RNA components (Figure 1), including mRNA, tRNA, and rRNA, while only bacterial mRNA can be recognized by mice NLRP3 inflammasome.⁸⁵ How NLRP3 inflammasome senses Salmonella RNA components, the contribution of each RNA component to the host immune response during Salmonella infection are significant questions that require further investigation. Flagellin, a recognized activator of NLRC4 inflammasome that can directly bind to NAIP, has been shown to activate the NLRP3 inflammasome in human macrophages in a NAIP-independent manner through the canonical pathway (Figure 1).⁸⁶ Whether this effect exists in mouse cells remains to be verified, and the mechanism of how NLRP3 inflammasome recognizes Salmonella flagellin warrants further exploration.

Several studies have revealed that the NLRP3 activation events are tied to alterations in host cell most likely induced by intracellular Salmonella metabolism. Specifically, excessive accumulation of bacterial citrate, an oxidative metabolite produced by Salmonella, can rapidly activate the NLRP3 inflammasome through an mtROS-dependent pathway (Figure 1), which is abrogated upon deletion of Salmonella citrate synthase GltA.³² Additionally, Salmonella disrupts glycolytic flux by consuming host-cell glucose, leading to the activation of NLRP3 inflammasome through sensing this metabolic shift. This activation is dependent on a reduction in NADH levels and the induction of mtROS production.⁸⁷ Our previous studies also support the notion that the NLRP3 inflammasome can sense intracellular Salmonella via metabolic pathways. We found that the bacterial DNA adenine methylase (Dam) is essential for the activation of the Jnk-dependent NLRP3 inflammasome during Salmonella infection (Figure 1).⁸⁸ Further research suggests that Dam is employed to repress the production and conversion of arachidonic acid to prostaglandin E2, mediated by cytosolic phospholipase A2 in macrophages.⁸⁹ These findings suggest that the innate immune responses mediated by the NLRP3 inflammasome can detect intracellular bacteria not only through sensing bacterial proteins and virulence activities but also by sensing bacterial metabolites.

In addition to the canonical NLRP3 inflammasome, the host innate immune system can recognize invading Salmonella through non-canonical and alternative NLRP3 pathways (Figure 1). Upon recognizing extracellular LPS of Salmonella, TLR4 induces the type I interferon response, along with the complement C3-C3aR axis, to promote Caspase-11 expression.⁹⁰ Type I interferons further induce the expression of guanylate-binding proteins (GBPs) and interferon-inducible protein IRGB10.⁹¹ Once recruited to the intracellular Salmonella cell membrane via their conserved transmembrane domains and myristoylation motifs, GBPs and IRGB10 form oligomeric complexes that disrupt bacterial membrane integrity. This process involves IRGB10 inserting its amphipathic helices into the bacterial membrane, leading to structural destabilization and subsequent release of LPS into the cytosol. The liberated LPS then binds directly to Caspase-11, triggering non-canonical activation of the NLRP3 inflammasome and pyroptosis.⁹² During the early stages of Salmonella infection, human monocytes can swiftly respond through the alternative NLRP3 inflammasome pathway, preceding the activation of the canonical, non-canonical NLRP3, or NLRC4 pathways.⁹³ The rapid activation of the alternative NLRP3 inflammasome may be dependent on TLR4 recognition of Salmonella LPS. Furthermore, early activation of alternative NLRP3 signaling may facilitate the subsequent triggering of canonical and non-canonical NLRP3 pathways, as the alternative pathway enhances the expression of NLRP3 and the release of IL-1β.

Strategies for Salmonella to Avoid NLRP3 Inflammasome Activation

The NLRP3 inflammasome swiftly responds to stimuli such as ATP, nigericin, or crystals within 1–2 hours,^94–96 resulting in rapid Caspase-1 activation and pro-inflammatory cytokines release. However, in bone marrow-derived macrophages (BMDMs) invaded by Salmonella, NLRP3 inflammasome activation is delayed, occurring only 12–16 hours post-infection.³⁰ The significance of this delayed activation of NLRP3 inflammasome during Salmonella infection remains controversial. The NLRP3 and NLRC4 inflammasome have been indicated to play redundant roles in resisting Salmonella infection.³⁰ Despite its role in cytokine maturation, NLRP3 and ASC have been reported to play a limited role during Salmonella infection.³¹ NLRP3 is dispensable for Salmonella clearance in the cecum and mesenteric lymph nodes, even in the absence of NLRC4.⁹⁷ Consistent with these findings, our previous research showed no significant difference in bacterial colonization and inflammasome-dependent cytokine secretion between WT- and Nlrp3^−/−-mice infected with Salmonella.³³ Although flagellin and SPI-1 T3SS apparatus of Salmonella are readily recognized by NLRC4 inflammasome within 1 hour of invasion, these PAMPs, which are crucial for bacterial invasion of epithelial cells, remain highly expressed during the logarithmic growth phase. Undeniably, NLRC4 inflammasome is the main force in recognition and clearance of intestinal Salmonella. The high activity of NLRC4 inflammasome indeed masks the role of NLRP3 inflammasome in the early stages of Salmonella infection. Based on in-depth studies, the delayed activation of NLRP3 inflammasome and its limited role in resisting Salmonella infection could be attributed to multiple strategies employed by Salmonella to avoid or delay NLRP3 activation during infection.

Once inside the host cell, Salmonella engages in an intracellular battle for survival with host cell molecular defense pathways. Salmonella strains isolated from mice during the acute phase of infection display an enhanced potential to activate the NLRP3 inflammasome. Conversely, Salmonella strains isolated during the chronic phase of infection exhibit a significantly attenuated ability to activate NLRP3 signaling and induce the secretion of IL-1β.⁹⁸ The findings suggest that Salmonella possesses the capability to adjust its NLRP3 inflammasome-activating potential across various infection stages, enabling it to adapt to the host intracellular environment. During chronic infections, Salmonella further adapts by evading NLRP3 inflammasome recognition, which aids in bacterial survival and the establishment of persistent infections. Concealing within Salmonella-containing vacuoles (SCVs) devoid of inflammasome activity provides Salmonella with a protective shield from detection. While this evasion strategy appears effective, it is not flawless. Long-term residence within the SCV hinders disease progression, some Salmonella will spontaneously escape from nascent SCV and replicate within the host cell cytosol.⁹⁹ Additionally, the SCV is constantly under attack by host immune defenses, which strive to destabilize it and induce its fusion with lysosomes.^21,22 SifA, a critical Salmonella SPI-2 T3SS effector, plays a vital role in maintaining the stability of the SCV (Figure 2). Salmonella deficient in SifA are rapidly released into the cell cytosol within a few hours after invading.^100,101 Early studies suggested that the enhanced clearance of sifA deletion mutants is mediated by Caspase-11-driven pyroptosis, a process independent of NLRP3, NLRC4, and ASC.²⁶ However, it is worth noting that Caspase-11-mediated pyroptosis is also sufficient to induce the activation of non-canonical NLRP3 inflammasome. Our recent high-throughput screenings further reveal that SifA plays a role in inhibiting the NLRP3 inflammasome activation in the absence of flagellin.³³ While it remains undetermined whether SifA suppresses the canonical or non-canonical NLRP3 pathway, the SCV stabilization mediated by SifA contributes to bacterial evasion of NLRP3 inflammasome activation.

Figure 2 Multiple strategies utilized by Salmonella to suppress NLRP3 inflammasome activation. Salmonella employs the PhoP-PhoQ and PmrA-PmrB two-component regulatory systems to chemically modify Lipopolysaccharide (LPS), thereby evading recognition by the non-canonical inflammasome. Furthermore, PhoP-PhoQ significantly down-regulates the expression of flagellin and T3SS-1 when Salmonella resides within macrophages. The T3SS effector proteins are enclosed within the circle marked by the red dashed line. The type III secretion system-2 (T3SS-2) effector protein SifA plays a crucial role in maintaining the stability of the Salmonella-containing vacuole (SCV), which is essential for Salmonella to prevent non-canonical NLRP3 inflammasome activation triggered by Caspase-4/5/11. The T3SS-2 effector SpvC inhibits NLRP3 inflammasome activation by inactivating dual-phosphorylated MAPK. Another T3SS-2 effector, SlrP, suppresses the NLRP3 inflammasome in myeloid cells within the lamina propria, thereby preventing anorexia caused by IL-1β-mediated signaling to the hypothalamus via the vagus nerve. The T3SS effector SteE is utilized by Salmonella to induce M2 macrophage polarization, fostering an environment with low NLRP3 activity. The T1SS protein SiiD, as well as bacterial tricarboxylic acid cycle (TCA) enzymes is essential for Salmonella to repress the mtROS-ASC-mediated NLRP3 inflammasome activation. Furthermore, UDP-galactose-4-epimerase GalE, the toxin and antitoxin system antitoxin protein DinJ, the fimbrial chaperone BcfB, the two-component system phosphate transferase RcsD, and the symporter of melibiose and monovalent cations MelB all exhibit inhibitory effects on NLRP3 inflammasome activation through undefined mechanisms. Figure 2 was independently designed by the authors using Microsoft PowerPoint 2013, with all elements created using native software features.

Once the SCV is breached, inflammasomes swiftly recognize Salmonella that have been released into the cytoplasm by detecting PAMPs such as LPS, flagellin, and SPI-1 T3SS. To counteract the recognition of these PAMPs, which are essential for bacterial invasiveness, Salmonella activates the PhoP-PhoQ regulatory system when surviving within macrophages.^102–104 This system potently represses the expression of flagellin and SPI-1 T3SS while concurrently enhancing the expression of SPI-2 T3SS.^105,106 By decreasing the expression of flagellin (Figure 2), Salmonella gains an advantage in evading canonical NLRP3 inflammasome activation, as NLRP3 has been found to sense flagellin in a NAIP-independent manner.⁸⁶

LPS is another target of the NLRP3 inflammasome induced by Salmonella. Intracellular LPS triggers non-canonical NLRP3 inflammasome activity via Caspase-4/5/11. In addition to down-regulation of PAMPs expression, Salmonella can also alter its detectable PAMPs by chemical modification. Specifically, Salmonella modifies its lipid A of LPS through the PhoP-PhoQ and PmrA-PmrB two-component regulatory systems (Figure 2).¹⁰⁷ Previous studies have shown that Caspase-11 exhibits reduced activity in response to tetra-acylated lipid IVa and penta-acylated LPS,^108,109 due to its inability to oligomerize once bound to these LPS forms. Notably, the LPS from intracellular WT Salmonella is hexa-acylated. The ability of Salmonella to stimulate IL-1β expression in human monocytes and infected mice is significantly reduced when LPS is modified into a penta-acylated form.¹¹⁰ Moreover, two additional structural modifications of LPS, LPS with a hepta-acylated lipid A and LPS with phosphoethanolamine and aminoarabinose attached to the phosphate groups of its lipid A, have been reported to induce reduced IL-1β production in human monocytes and mouse macrophages.^111,112 Additionally, the LPS O‐antigen length regulator FepE has been found to induce the production of very long O‐antigen chains, which are crucial for Salmonella to evade recognition by Caspase-4 and subsequent non-canonical NLRP3 inflammasome activation.¹¹³ The secretion of IL-1β promoted by activated Caspase-4/5/11 relies on the non-canonical NLRP3 signaling pathway, suggesting that diverse chemical modifications of LPS serve as an effective strategy for Salmonella to evade NLRP3 inflammasome activity and consequently promote its intracellular survival.

In addition to modulating PAMPs to avoid detection by the NLRP3 inflammasome, Salmonella employs multiple bacterial virulence factors to inhibit its activation by targeting the NLRP3 signaling pathway. Our recent study has identified a novel virulence factor in Salmonella that functions in anti-inflammatory responses during infection. Specifically, the T1SS protein SiiD, which is translocated into host cells in a T1SS-dependent manner and localized in the membrane fraction, is utilized by Salmonella to suppress the activation of the intracellular canonical NLRP3 inflammasome (Figure 2). Mechanistically, SiiD suppresses the generation of intracellular mtROS, which subsequently prevents ASC oligomerization from forming pyroptosomes, resulting in the inhibition of NLRP3-dependent Caspase-1 activation and the secretion of IL-1β and IL-18.³³ Importantly, SiiD is required for Salmonella virulence, enabling the bacterium to evade host immune clearance mediated by the NLRP3 inflammasome in vivo, which may be crucial for bacterial colonization and persistence during long-term infection. These findings reveal the essential role of bacterial T1SS in inhibiting NLRP3 inflammasome activation and enabling Salmonella to evade host innate immune defenses. Beyond T1SS, SPI-2 encoded T3SS-2 also plays a pivotal role in the intracellular replication and survival of Salmonella. The T3SS-2 effector SpvC is utilized by Salmonella to repress NLRP3 inflammasome activation,¹¹⁴ potentially through the inactivation of dual-phosphorylated MAPK via beta elimination (Figure 2).¹¹⁵ SpvC has also been verified to suppress pyroptosis and intestinal inflammation in the cecum via its phosphothreonine lyase activity, which is necessary for promoting bacterial dissemination.¹¹⁴ Furthermore, a recent study has revealed that SPI-2 is employed by Salmonella to inhibit NLRP3 inflammasome activation in human macrophages, but not in mouse macrophages.^116,117 The mechanism by which SPI-2 inhibits the NLRP3 inflammasome activation in human cells, whether through the secretion of T3SS effector proteins or the regulation of other signaling pathways, remains to be elucidated by further research.

A prior study has demonstrated that three additional virulence factors of Salmonella, the fimbrial chaperone BcfB, the two-component system phosphate transferase RcsD, and the symporter of melibiose and monovalent cations MelB, all possess the capability to interfere with the activation of the NLRP3 inflammasome via unknown mechanisms (Figure 2).³² Our recent high-throughput screening has further confirmed the role of the Rcs two-component system in regulating NLRP3 inflammasome activation.³³ Additionally, our previous research demonstrated that the bacterial toxin and antitoxin system antitoxin protein DinJ can be transported into host cells during Salmonella infection (Figure 2), which is crucial for Salmonella to specifically inhibit NLRP3 inflammasome activation and evade the immune clearance mechanisms mediated by NLRP3 in vivo.¹¹⁸ However, it is worth noting that the inhibition of NLRP3 inflammasome activation mediated by Salmonella is not always beneficial to bacterial virulence. For instance, the Salmonella T3SS-2 effector protein SlrP inhibits the NLRP3 inflammasome in myeloid cells in the lamina propria, preventing anorexia caused by IL-1β-mediated signaling to the hypothalamus via the vagus nerve (Figure 2).¹¹⁹ Mice orally infected with ΔslrP exhibited a more severe anorexic response, along with increased pathogen burdens and higher mortality, because of a significant increase in the expression of appetite-suppressing genes in the hypothalamus.³⁴ SlrP-mediated inhibition of the NLRP3 inflammasome thus prevents anorexia induced by Salmonella infection, promoting the survival of both the pathogen and the host. This mechanism is essential for the bacterial transmission to new hosts, albeit at the expense of virulence. These findings indicate that Salmonella has evolved a sophisticated strategy to balance transmission and virulence by specifically targeting the NLRP3 inflammasome.

The ability of bacteria to sense metabolic alterations within host cells constitutes another crucial strategy for circumventing the detection of the NLRP3 inflammasome. Previous studies have demonstrated that Salmonella preferentially survives within macrophages that display an M2 phenotype, which primarily utilize oxidative metabolism rather than glycolysis.^120,121 Salmonella infection of M1 macrophages results in the consumption of glycolytic precursor molecules, which are critical energy sources for host cells. This disruption of glycolysis subsequently triggers robust activation of the intracellular NLRP3 inflammasome.⁸⁷ In contrast, Salmonella-infected M2 macrophages exhibit upregulated expression of the transcription factor PPARδ. This transcription factor plays a pivotal role in maintaining fatty acid β-oxidation metabolism. Mechanistically, PPARδ directly enhances glucose availability to intracellular Salmonella, thereby promoting bacterial replication.¹²⁰ Upon induction of the M1-to-M2 polarization shift via medication, a significant reduction in Caspase-1 activity, accompanied by decreased expression levels of NLRP3, IL-1β, and IL-18, was observed.¹²² Another study reported an increase in the number of M2 macrophages in NLRP3-deficient mice.¹²³ A recent study reports that the SPI-2 T3SS effector SteE is employed by Salmonella to convert both the amino acid and substrate specificity of the host pleiotropic serine/threonine kinase GSK3, which enables GSK3 to phosphorylate a tyrosine residue on the non-canonical substrate STAT3, ultimately driving M2 macrophage polarization (Figure 2).¹²⁴ Compared to mouse BMDMs infected with WT Salmonella strain, cells infected with ΔsteE mutant show diminished M2 polarization along with reduced phosphorylated-STAT3 level.¹²⁵ Another recent study reveals that Salmonella infection triggers macrophages to produce lactate, resulting in enhanced translocation of the SPI-2 effector SteE, which is essential for driving subsequent M2 macrophage polarization.¹²⁶ These findings suggest that Salmonella not only possesses the capability to target cellular niches exhibiting reduced NLRP3 inflammasome activity by sensing metabolic changes within host cells, but also promotes M2 macrophage polarization through the translocation of T3SS effectors to establish a favorable cellular niche for bacterial replication and persistence.

Salmonella tricarboxylic acid cycle (TCA) enzymes, including aconitase AcnB, isocitrate lyase AceA, and isocitrate dehydrogenase IcdA, are crucial for inhibiting the rapid NLRC4-independent NLRP3 inflammasome activation during the early stages of infection (Figure 2). Deletion of TCA cycle enzymes leads to excessive citrate production in Salmonella-infected cells, which in turn triggers rapid mtROS-dependent NLRP3 inflammasome activation. Furthermore, aconitase-deficient Salmonella demonstrated a notable impairment in acute systemic virulence after oral administration and exhibited a reduced capacity to persist in a chronic infection.³² In addition, our recent study has shown that the Salmonella UDP-galactose-4-epimerase GalE, which catalyzes the interconversion between UDP-glucose and UDP-galactose, is involved in inhibiting the NLRP3 inflammasome activation and IL-1β expression within host macrophages (Figure 2).¹²⁷ This effect may depend on regulation of multiple inflammasome-related signaling pathways mediated by GalE and is crucial for bacterial virulence. These findings indicate that metabolic alterations serve as a pivotal target in the complex interplay of detection and evasion between Salmonella and the NLRP3 inflammasome.

Conclusion

With the relentless pursuit of research delving deeper into the intricate interplay between Salmonella and the NLRP3 inflammasome, the highly sophisticated molecular mechanisms driving NLRP3 inflammasome activation, as well as crucial mechanisms that enable the host to restrict the proliferation of this intracellular pathogen, have been progressively unveiled. The NLRP3 inflammasome signaling is dynamically modulated during the chronic infection of Salmonella. The delicate balance between pathogen proliferation and host immune defenses fosters a mutually beneficial coexistence for both organisms. Pathogens that exhibit high virulence will kill hosts, whereas those with reduced virulence may achieve persistent infection. The key strategy for chronic survival of the intracellular bacteria is to down-regulate their ability to trigger the NLRP3 inflammasome signaling. As the structural mechanisms underlying NLRP3 inflammasome assembly are progressively elucidated, future research endeavors will increasingly focus on elucidating the intricate structural details of ligand-sensor interactions. Identifying more ultimate NLRP3 ligands or determining the minimal common cellular event that is both necessary and sufficient for activating the NLRP3 inflammasome during Salmonella infection will be a significant achievement. Furthermore, despite a growing body of research, our understanding of the molecular mechanisms by which Salmonella evades or inhibits the activation of NLRP3 inflammasome remains incomplete. Future research endeavors are necessary to identify the key Salmonella molecules that possess the ability to potently impede or facilitate NLRP3 inflammasome assembly or activation. Targeting these pivotal bacterial molecules for the design of therapeutic drugs may emerge as a promising strategy, contributing to the advancement of novel treatments and preventive measures for salmonellosis.