Macrophage migration inhibitory factor of zoonotic Giardia duodenalis triggers TLR4-MAPK/AKT/NLRP3 pathways to regulate inflammatory response

Summary

Giardia duodenalis is an important zoonotic protozoan that has a significant negative impact on public health worldwide. G. duodenalis macrophage migration inhibitory factor (GdMIF) has close similarity with mammalian MIF, yet its immunomodulatory role remains unclear. This study demonstrated that G. duodenalis secretes GdMIF, which functions as a potent immunostimulator in mouse peritoneal macrophages. Recombinant GdMIF (rGdMIF) upregulated TLR2/4 expression, induced robust cytokines secretion, and activated the MAPK, AKT, and NF-κB signaling pathways. Mechanistically, rGdMIF activated MAPK and AKT pathways via TLR4, but not TLR2. TLR4 deficiency (TLR4^−/−) impaired cytokines production and reduced phosphorylation of p38, ERK, and AKT. Inhibition of p38 or ERK further suppressed cytokines release, while AKT inhibition slightly enhanced cytokines production, whereas TLR2^−/− had no effect. Moreover, rGdMIF activated the NLRP3 inflammasome in a TLR4- and NLRP3-dependent manner. These findings demonstrated that GdMIF is a novel G. duodenalis-derived pathogen-associated molecular pattern (PAMP) recognized by TLR4, providing new insights into host-parasite immune interactions.

Graphical abstract

Highlights

• •GdMIF is a novel G. duodenalis PAMP recognized by TLR4
• •GdMIF triggers inflammatory cytokine release via TLR4-MAPK/AKT pathways
• •TLR4 mediates GdMIF-induced NLRP3 inflammasome activation and IL-1β release

Immunology; microbiology; cell biology

Introduction

Giardia duodenalis (G. duodenalis) is a globally distributed zoonotic protozoan transmitted through ingestion of cysts in contaminated food or water, causing giardiasis in humans and diverse mammalian hosts.¹ This infection poses a substantial public health burden, with an estimated 280 million human cases annually.² While often asymptomatic, symptomatic giardiasis manifests as vomiting, abdominal pain, weight loss, severe diarrhea, and malabsorption syndrome, particularly affecting travelers to endemic regions, children, and individuals exposed to untreated water.^3,4 Current treatment relies primarily on metronidazole and tinidazole.⁴ However, the drug resistance of these anti-giardial agents has become increasingly common in recent years, carries the risk of teratogenicity and mutation.⁵ Currently, studies have identified G. duodenalis proteins such as α-enolase, ornithine carbamoyltransferase, and variant surface proteins (VSP9B10, VSP1267, and VSPH7) can induce protective immune responses and are promising candidates for further vaccine research,^6,7 but there is currently no commercialized vaccine against G. duodenalis. Hence, there is a pressing need to identify novel therapeutic targets for the prevention and treatment of giardiasis.

Pathogen infection is fought first by innate immune responses. Pattern-recognition receptors (PRRs) detect pathogen-associated molecular patterns (PAMPs), triggering the production of antimicrobial peptides, cytokines, and chemokines. This response serves to limit initial pathogen replication and subsequently orchestrates adaptive immunity, ultimately enhancing host resistance.^8,9,10,11 Both innate and adaptive mechanisms contribute to controlling G. duodenalis infection.¹² Key PRR families include Toll-like receptors (TLRs), RIG-I-like receptors (RLRs), NOD-like receptors (NLRs), and cytosolic DNA sensors. Evidence implicates several PRRs in sensing G. duodenalis. TLRs are crucial innate immune signaling receptors.¹³ Studies have found that G. duodenalis and its extracellular vesicles activate p38 and AKT signaling pathways via TLR2, regulate proinflammatory cytokines secretion in murine peritoneal macrophages (PMϕs).^14,15 Furthermore, G. duodenalis binding immunoglobulin protein engages TLR4, initiating the MyD88-p38/extracellular signal-regulated kinase (ERK) signaling cascade to induce dendritic cell maturation.¹⁶ The NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome, a component of the NLR family,⁹ is also activated by G. duodenalis, its extracellular vesicles, and G. duodenalis protein peptidyl-prolyl cis-trans isomerase B (PPIB) leading to IL-1β production.^14,17 Crucially, G. duodenalis triggers NLRP3 inflammasome activation in macrophages in a TLR4-dependent manner.¹⁸ Additionally, G. duodenalis and its excretory-secretory products (ESPs) activate the NOD2-Rip2-ROS axis to modulate proinflammatory cytokine (IL-6, IL-12, and TNF-α) production in macrophages.¹⁹ G. duodenalis infection induces the production of cytokines such as IL-1β, IL-6, IL-12, and TNF-α, which mediate critical host defenses against the parasite.^12,20 Despite this progress, the specific G. duodenalis molecules recognized by TLRs or NLRs remain largely unexplored. To date, only two examples are characterized: G. duodenalis arginine deiminase induces IL-1β, IL-6, and TNF-α secretion via TLR2 and TLR4 interaction.²¹ G. duodenalis PPIB activates NLRP3 inflammasome and pyroptosis via TLR4.¹⁸ Consequently, identifying additional Giardia-derived immunomodulatory components and their mechanisms is essential for developing novel therapeutic strategies against giardiasis.

Macrophage migration inhibitory factor (MIF) is initially identified as a lymphocytosis produced by activated T lymphocytes to inhibit the random migration of macrophages.²² MIF is located on chromosome 22 of the human genome and encodes a 114-amino acid nonglycosylation protein that exhibits ∼90% close similarity in mammals.^23,24 The function of mammalian MIF is found to be involved in innate immune responses and plays a key role in triggering inflammatory responses.²⁵ Interestingly, several protozoa, such as Entamoeba histolytica, Plasmodium falciparum, Leishmania, Trichomonas vaginalis, and Toxoplasma gondii express MIF homologs capable of modulating host immune responses.^26,27,28 For instance, immunization with T. gondii MIF elicits a robust immune response characterized by high IFN-γ and IgG levels, correlating with reduced parasite cyst burden.²⁹ T. vaginalis MIF activates ERK and AKT pathways, promoting IL-8 secretion in human prostate cells and potentially contributing to cellular proliferation and carcinogenesis.²⁸ Current research on G. duodenalis MIF (GdMIF) has identified a gene encoding a 114-amino acid protein with a predicted 12.5 kDa molecular weight. Its crystal structure reveals a characteristic α/β fold, formed by a four-stranded β-sheet against which two antiparallel α-helices are packed. The assembly of a functional trimer is stabilized by an additional short β-strand from an adjacent subunit, which aligns antiparallel to the β4 strand, contributing to a discontinuous β-barrel architecture.³⁰ GdMIF and humans (HsMIF) and three Plasmodium species (falciparum, berghei, and yoelii) MIF are in close similarity.³⁰ However, its role in regulating host immune responses and the underlying mechanisms remain undefined.

In this study, we investigated the capacity of rGdMIF to activate TLRs and NLRs, thereby regulating cytokine production. The effects of rGdMIF on TLR2, TLR4, MAPK, AKT, NF-κB pathway, and the secretion of cytokines (IL-6, IL-10, TNF-α, IL-12, and IL-1β) were examined in rGdMIF-stimulated mouse PMϕs. The regulatory role of rGdMIF in cytokine induction was further verified by using TLR2-deficient (TLR2^−/−) and TLR4^−/− mouse PMϕs, molecular docking, and specific pharmacological inhibitors of MAPK and AKT. Concurrently, NLRP3 inflammasome activation (measured by canonical indicators) and IL-1β secretion were detected in rGdMIF-stimulated mouse PMϕs, and NLRP3^−/− mouse PMϕs were used to verify NLRP3 inflammasome activation. Furthermore, to elucidate potential crosstalk between pathways, TLR2 and TLR4^−/− mouse PMϕs used to explore the roles of TLRs in rGdMIF-induced NLRP3 inflammasome activation.

Results

rGdMIF regulated the secretion of inflammatory cytokines via TLR4 in mouse PMϕs

Multiple pathogens can secrete MIF protein to regulate the host’s immune response, such as T. gondii, T. vaginalis, P. falciparum, and Mycobacterium tuberculosis. Therefore, to detect whether G. duodenalis also secretes GdMIF, the ESPs of G. duodenalis were collected and used to measure GdMIF. The result showed that the GdMIF protein was present in GdESP (Figure 1A). To understand the role of rGdMIF on inducing inflammatory response, we purified and obtained rGdMIF that was tag-free (Figure S1A) and the content of endotoxin is 0.03 EU/mL (Figure S1B), and mouse PMϕs were stimulated with rGdMIF, the levels of inflammatory cytokines secretion were detected by ELISA, and TLRs expression were investigated by qPCR. The results showed that rGdMIF stimulation significantly increased the secretion of IL-6 (Figure 1B), IL-10 (Figure 1C), IL-12 p40 (Figure 1D), TNF-α (Figure 1E), and IL-1β (Figure 1F) in a dose-dependent manner in WT PMϕs. rGdMIF up-regulated the mRNA and protein levels of TLR2 and TLR4 in WT PMϕs (Figures 1G and 1H). To determine the specific TLR involved, we compared the levels of cytokines secretion in rGdMIF-stimulated WT, TLR2^−/−, and TLR4^−/− mouse PMϕs. rGdMIF-induced the secretion levels of IL-6 (p < 0.0001) (Figure 1I), IL-10 (p < 0.001) (Figure 1J), IL-12 p40 (p < 0.0001) (Figure 1K), TNF-α (p < 0.05) (Figure 1L), and IL-1β (p < 0.05) (Figure 1M) were significantly reduced in TLR4^−/− PMϕs compared to WT PMϕs, whereas were not changed in TLR2^−/− PMϕs (p > 0.05) (Figures 1I–1M). Molecular docking analysis indicated that GdMIF formed a stable interaction with TLR4 (Figure 1N). These results suggested that rGdMIF regulated the secretion of inflammatory cytokines via TLR4 signal pathway.

Figure 1.

rGdMIF regulated inflammatory cytokines secretion via TLR4 activation

(A) GdMIF protein in GdESP was detected by western blot.

(B–F) The secretion levels of cytokines were measured by ELISA. Gd stands for G. duodenalis.

(G) WT mouse PMϕs were treated with PBS, rGdMIF (1 μg/mL), and G. duodenalis trophozoites (3 × 10⁶) for 2 h, the transcription levels of TLR2, TLR3, and TLR4 were detected using qPCR.

(H) PMϕs were stimulated with rGdMIF at different times or concentrations, the expression levels of TLR2 and TLR4 proteins were detected by western blot.

(I–M) WT, TLR2^−/− and TLR4^−/− PMϕs were stimulated with rGdMIF for 24 h, and cell culture supernatants were collected. The secretion levels of cytokines were measured by ELISA.

(N) The molecular docking model of GdMIF (yellow) with TLR4 (blue) protein. Data are presented as mean ± SD from three independent experiments (n = 3), statistical significance was assessed using one-way ANOVA. n.s. (p > 0.05) indicates not significant, and ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

rGdMIF mediated the activation of MAPK and AKT pathways via TLR4

The phosphorylation levels of p38, ERK, AKT, p65, and IκB in rGdMIF-stimulated mouse PMϕs were detected using western blot. Significantly increased phosphorylation of p38 (p < 0.01) (Figures 2A and 2B), ERK (p < 0.01) (Figures 2A and 2C), AKT (p < 0.0001) (Figures 2A and 2D), p65 (p < 0.05) (Figures 2E and 2F), and IκB (p < 0.05) (Figures 2E and 2G) was observed in WT PMϕs stimulated with rGdMIF for 2 h compared to the unstimulated control group (NC group). In dose-response experiments, WT PMϕs stimulated with increasing rGdMIF concentrations for 2 h showed significantly elevated phosphorylation of p38 (p < 0.05) (Figures 2H and 2I), ERK (p < 0.05) (Figures 2H and 2J), p65 (p < 0.05) (Figures 2L and 2M), and IκB (p < 0.01) (Figures 2L and 2N) starting at 0.5 μg/mL. AKT phosphorylation (p < 0.01) (Figures 2H and 2K) was significantly increased at ≥1 μg/mL compared to the NC group, therefore, PMϕs were stimulated with 1 μg/mL rGdMIF in the subsequent study.

Figure 2.

rGdMIF activates MAPK, AKT, and NF-κB signaling pathway in mouse PMϕs

(A and E) PMϕs were stimulated with rGdMIF (1 μg/mL) at different times, the expression of total and phosphorylated proteins of p38, ERK, AKT, p65, and IκB were detected by western blot.

(B–D, F, and G) The expression level of protein was analyzed by ImageJ.

(H and L) PMϕs were stimulated with different concentration rGdMIF for 2 h, the expression of total and phosphorylated proteins of p38, ERK, AKT, p65, and IκB were detected by western blot.

(I–K, M, and N) The expression level of protein was analyzed by ImageJ. Data are presented as mean ± SD from three independent experiments (n = 3), statistical significance was assessed using one-way ANOVA. n.s. (p > 0.05) indicates not significant, and ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

To investigate whether rGdMIF activated MAPK, AKT, and NF-κB pathways via TLR2 or TLR4, WT, TLR2^−/− and TLR4^−/− PMϕs were stimulated with 1 μg/mL rGdMIF for 2 h. Western blot results showed that rGdMIF-stimulated the phosphorylation levels of p38 (p < 0.001) (Figures 3A and 3B), ERK (p < 0.001) (Figures 3A and 3C), and AKT (p < 0.001) (Figures 3A and 3D) were significantly attenuated in TLR4^−/− PMϕs compared to WT PMϕs, whereas were not significant changed in TLR2^−/− PMϕs (p > 0.05) (Figures 3A–3D). The phosphorylation levels of p65 (p > 0.05) (Figures 3E and 3F) and IκB (p > 0.05) (Figures 3E and 3G) showed no significant differences between TLR2^−/−, TLR4^−/− and WT PMϕs. These results suggested that rGdMIF activated MAPK and AKT pathways via TLR4.

Figure 3.

Roles of TLR4 receptor in rGdMIF triggered MAPK, AKT, and NF-κB signaling pathway

WT, TLR2^−/− and TLR4^−/− PMϕs were stimulated with rGdMIF (1 μg/mL) for 2 h, respectively.

(A and E) The expression of total and phosphorylated p38, ERK, AKT, p65, and IκB were detected by western blot.

(B–D, F, and G) The expression level of protein was analyzed by ImageJ. Data are presented as mean ± SD from three independent experiments (n = 3), statistical significance was assessed using one-way ANOVA. n.s. (p > 0.05) indicates not significant, ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

rGdMIF induced inflammatory cytokines production via MAPK and AKT pathways

To investigate whether rGdMIF induced inflammatory cytokines secretion via p38, ERK or AKT signaling pathway, PMϕs were pretreated with p38 inhibitor (SB203580), ERK inhibitor (PD98059), or AKT inhibitor (MK-2206 2HCl) and stimulated with rGdMIF. Cells and culture supernatants were collected for western blot and ELISA, respectively. The results showed that inhibitor pretreatment significantly reduced rGdMIF-induced phosphorylation of p38 (p < 0.0001) (Figure 4A), ERK (p < 0.0001) (Figure 4B), and AKT (p < 0.0001) (Figure 4C) compared to untreated PMϕs. Furthermore, pretreatment with SB203580 (p38 inhibitor) or PD98059 (ERK inhibitor) significantly decreased rGdMIF-induced production of IL-6 (p < 0.001, p < 0.001) (Figure 4D), IL-10 (p < 0.05, p < 0.01) (Figure 4E), IL-12 p40 (p < 0.001, p < 0.0001) (Figure 4F), TNF-α (p < 0.001, p < 0.001) (Figure 4G), and IL-1β (p < 0.001, p < 0.001) (Figure 4H), and pretreatment with MK-2206 2HCl (AKT inhibitor) increased rGdMIF-induced TNF-α production (p < 0.05) (Figure 4G). Collectively, these results indicated that rGdMIF induced inflammatory cytokines secretion primarily through p38 and ERK signaling pathways, while AKT signaling exhibited a divergent regulatory effect on TNF-α.

Figure 4.

rGdMIF regulated inflammatory cytokines secretion through the p38, ERK, and AKT signaling pathways

WT mouse PMϕs were pretreated with SB203580 (30 μM), PD98059 (40 μM), or MK-2206 2HCl (5 μM) and stimulated with rGdMIF (1 μg/mL).

(A–C) The phosphorylation levels of p38, ERK, and AKT proteins were detected by western blot, and the expression levels of proteins were analyzed by ImageJ. Data are displayed as mean ± SD, statistical significance was assessed using unpaired Student’s t test.

(D–H) The productions of cytokines were measured by ELISA. Data are presented as mean ± SD from three independent experiments (n = 3), statistical significance was assessed using one-way ANOVA. n.s. (p > 0.05) indicates not significant, and ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

rGdMIF induced NLRP3 inflammasome activation in mouse PMϕs

To determine whether rGdMIF induced inflammasome activation, we used rGdMIF to stimulate LPS-pretreated PMϕs to detect NLRP3 inflammasome activation at time courses and concentration gradients. Western blot results showed that rGdMIF stimulation caused NLRP3 protein expression, and the cleavages of Caspase-1 and IL-1β (Caspase-1 p20 and IL-1β p17) were significantly increased (Figures 5A and 5D), and the releases of IL-1β (p < 0.001) (Figures 5B and 5E) and LDH (p < 0.001) (Figures 5C and 5F) were increased in time and concentration dependent compared to NC group. IFA demonstrated that rGdMIF stimulation induced NLRP3 aggregation, a pattern mirroring that observed in the G. duodenalis group and the ATP group (Figure 6A).

Figure 5.

Activation of NLRP3 inflammasome in rGdMIF-stimulated mouse PMϕs

LPS-pretreated WT PMϕs were incubated with rGdMIF for 6 h, 12 h, and 24 h.

(A) NLRP3 inflammasome-related proteins expression was detected by western blot.

(B) The production of IL-1β was measured by ELISA.

(C) The amount of cell death was measured by LDH. 10 ng/mL, 0.5, 1, 5, 10, and 20 μg/mL rGdMIF incubated LPS-pretreated PMϕs for 24 h.

(D) NLRP3 inflammasome-related proteins expression was detected by western blot.

(E) The production of IL-1β was measured by ELISA.

(F) The amount of cell death was measured by LDH. Data are presented as mean ± SD from three independent experiments (n = 3), statistical significance was assessed using one-way ANOVA. n.s. (p > 0.05) indicates not significant, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.

Figure 6.

Roles of TLR4 and NLRP3 receptors in rGdMIF triggered inflammatory response

(A) LPS-pretreated WT PMϕs were stimulated with rGdMIF (1 μg/mL) for 24 h, NLRP3 protein in PMϕs was observed by IFA. Blue: nuclei, Red: NLRP3 protein. Scale bars, 5 μm.

(B and E) LPS-pretreated WT, NLRP3^−/−, TLR2^−/− and TLR4^−/− mice PMϕs were stimulated with rGdMIF (1 μg/mL) for 24 h, respectively. NLRP3 inflammasome-related protein expressions were detected using western blot.

(C and F) The production of IL-1β was measured by ELISA.

(D and G) The amount of cell death was measured by LDH. Data are presented as mean ± SD from three independent experiments (n = 3), statistical significance was assessed using one-way ANOVA. n.s. (p > 0.05) indicates not significant, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.

Subsequently, LPS-pretreated WT and NLRP3^−/− PMϕs were stimulated with rGdMIF for 24 h, respectively. Western blot analysis revealed that IL-1β, Caspase-1 p20, and IL-1β p17 protein levels were significantly decreased in the rGdMIF-stimulated NLRP3^−/− PMϕs group compared to the WT PMϕs group (Figure 6B). Consistently, the release of IL-1β (p < 0.0001) (Figure 6C) and LDH (p < 0.0001) (Figure 6D) was also significantly reduced in the rGdMIF-stimulated NLRP3^−/− PMϕs group relative to the WT PMϕs group. These results suggested that rGdMIF induced NLRP3 inflammasome activation to secrete IL-1β in mouse PMϕs.

rGdMIF regulated NLRP3 inflammasome activation via TLR4 in mouse PMϕs

Further to determine whether TLR2 or TLR4 regulated rGdMIF-induced NLRP3 inflammasome activation, LPS-pretreated WT, TLR2^−/− and TLR4^−/− PMϕs were stimulated with rGdMIF for 24 h, respectively. Western blot analysis revealed significantly reduced protein levels of NLRP3 and pro-IL-1β, along with diminished cleavage of Caspase-1 p20 and IL-1β p17 in the rGdMIF-stimulated TLR4^−/− PMϕs group compared to the WT PMϕs group, but no significant differences were detected between TLR2^−/− and WT PMϕs (Figure 6E). The releases of IL-1β (p < 0.0001) (Figure 6F) and LDH (p < 0.0001) (Figure 6G) were also significantly reduced in the rGdMIF-stimulated TLR4^−/− PMϕs group compared to the WT PMϕs group. TLR2 deficiency did not affected the release of IL-1β (p > 0.05) (Figure 6F) and LDH (p > 0.05) (Figure 6G) compared to the WT PMϕs group. Collectively, these results indicated that rGdMIF activated the NLRP3 inflammasome to drive IL-1β secretion in a TLR4-dependent manner.

Discussion

Mammalian MIF plays a well-established role in immune responses, promoting the secretion of diverse cytokines.³¹ An emerging paradigm reveals that a variety of pathogenic protozoa can secrete MIFs, which can directly cause inflammatory cytokine secretion.³² For instance, Entamoeba histolytica MIF induces the secretion of IL-6 and TNF-α.²⁷ T. gondii MIF promotes the production of IFN-γ and IgG antibodies.²⁹ P. yoelii MIF up-regulates IFN-γ and other cytokines in mice.²⁶ T. vaginalis MIF promotes IL-8 secretion.²⁸ Previous studies showed that G. duodenalis and GdESP induce proinflammatory cytokines secretion.^33,34 Our findings align with this pattern, demonstrating that rGdMIF potently induced the secretion of IL-6, IL-12, TNF-α, and IL-1β in mouse PMϕs. Critically, IL-6 and TNF-α are known effector cytokines capable of mediating parasite clearance in G. duodenalis infection models.^35,36 This suggests that GdMIF, by stimulating these cytokines, may paradoxically contribute to host defense mechanisms against giardiasis, highlighting its complex role in host-parasite interactions.

TLRs are immune regulators and immunosurveillance receptors, trigger cells to release a number of inflammatory cytokines.³⁷ Lipopolysaccharide induces MIF expression in fibroblasts, and MIF regulates TLR4 to mediate inflammatory factor production.³⁸ However, the relationship between parasite-derived MIF and TLRs has not been reported. Previous work showed that G. duodenalis-treated PMϕs significantly upregulate TLR2 and TLR4 expression,^14,18 and G. duodenalis induced the secretion of TNF-α, IL-12, IL-10, and IL-6 via TLR2 or TLR4 activation.^14,39 In our study, we found rGdMIF upregulated the expression levels of TLR2 and TLR4, and the secretion levels of IL-6, IL-10, IL-12 p40, TNF-α, and IL-1β induced by rGdMIF were significantly reduced in TLR4-deficient PMϕs. However, TLR2 deficiency did not alter these cytokines production, revealing a differential role for these receptors and establishing that rGdMIF stimulates a TLR4-dependent, TLR2-independent inflammatory response. Our results provide the first evidence linking a protozoan MIF to TLR4 signaling, consistent with the immune response induced by G. duodenalis-infected mouse PMϕs.

Pathogen modulation of host innate immunity is often initiated by the interaction between PAMPs and PRRs. For instance, G. duodenalis VSPAS7 interacts with NLRP3 to regulate macrophage pyroptosis,⁴⁰ while Akkermansia muciniphila’s filamentous membrane protein Amuc-1100 interacts with TLR4 to modulate immune responses mediated by colonic RORγt⁺ Treg cells.⁴¹ In light of our finding that rGdMIF activated a TLR4-dependent pathway, we sought to elucidate the potential regulation mechanism. We performed a Z-DOCK computational docking simulation between GdMIF and TLR4, revealing the most likely docking mode, which indicated that GdMIF and TLR4 could bind to each other. Therefore, we speculated that GdMIF as a novel G. duodenalis PAMP, is recognized by TLR4 to initiate innate immune responses.

MIFs activate inflammatory effector proteins by switching on a variety of receptor-mediated signaling pathways. P. yoelii MIF activates the host cells MAPK-ERK1/2 signal pathway.⁴² Additionally, human MIF induces the secretion of TNF-α, IL-6, IL-8, and IL-12 cytokines through AKT and NF-κB pathway.^43,44 Our data demonstrated that rGdMIF rapidly activated p38, ERK1/2, AKT, and NF-κB signaling pathways in PMϕs. This aligns with reports that G. duodenalis and G. duodenalis ES products also induced proinflammatory cytokines production by activating p38, ERK1/2 pathways, and inhibited inflammatory cytokines production via AKT signaling pathway.^33,34,35 Crucially, TLR4 deficiency significantly attenuated rGdMIF-induced phosphorylation of p38, ERK, and AKT, but not NF-κB p65 or IκBα. Furthermore, the deficiency of TLR2 had no effect on the phosphorylation of p38, ERK, AKT, NF-κB p65, and IκBα. This identifies p38, ERK, and AKT as major downstream pathways activated by TLR4 upon GdMIF recognition. Pharmacological inhibition further confirmed that p38 and ERK pathways are essential for rGdMIF-induced secretion of IL-6, IL-10, IL-12, TNF-α, and IL-1β, while AKT inhibition unexpectedly enhanced TNF-α production, suggesting complex regulatory crosstalk. This divergent role of AKT mirrors findings with G. duodenalis, where AKT can negatively regulate cytokine production,⁴² underscoring the context-dependent nature of AKT signaling in giardial immunomodulation.

Activation of NLRP3 inflammasome is involved in host defense against pathogens through secretion of IL-1β.⁴⁵ Previous studies have reported that multiple protozoa infected cells induced NLRP3 inflammasome activation, such as Neospora caninum, T. gondii, Leishmania, and Entamoeba.^45,46 Recent studies have demonstrated that G. duodenalis trophozoites and G. duodenalis EVs activated NLRP3 inflammasome in mouse macrophages.^14,18 We extend this knowledge by demonstrating that rGdMIF induced the activation of NLRP3 inflammasome, evidenced by increased NLRP3 protein expression, Caspase-1 cleavage (p20), IL-1β (p17), IL-1β secretion, LDH release, and cytoplasmic NLRP3 aggregation. This response was abrogated in NLRP3^−/− PMϕs, confirming NLRP3 dependency. TLRs can prime or directly facilitate NLRP3 activation. Precedent exists for TLRs involvement, such as TLR2 promoting NLRP3 activation and IL-1β production in Prevotella nigrescens-infected dendritic cells,⁴⁷ and TLR2 deletion inhibits NLRP3 activation in allergic inflammation.⁴⁸ Notably, a direct mechanistic link between TLR4 and NLRP3 activation by G. duodenalis has been established: G. duodenalis induces macrophage NLRP3 inflammasome activation via TLR4/ROS signaling.¹⁸ Our data provided compelling evidence that GdMIF utilizes this same TLR4-dependent pathway, stimulation of TLR4^−/− macrophages with rGdMIF resulted in significantly attenuated NLRP3 protein levels, diminished Caspase-1 p20 and IL-1β p17 cleavage, decreased IL-1β secretion, and lower LDH release compared to wild-type cells, but these indicators of NLRP3 inflammasome activation showed no significant differences between TLR2^−/− and WT PMϕs. This robustly positions TLR4 as the primary receptor mediating GdMIF-induced NLRP3 inflammasome activation. While TLR4 emerges as critical for GdMIF signaling, the potential contribution of other TLRs or PRRs to this process remains an open question worthy of future investigation.

The functional consequence of TLR4/NLRP3 pathway activation is believed to be enhanced pathogen control. Cytokines like IL-1β, produced downstream of NLRP3 activation, along with IL-6, TNF-α, and IL-12 induced via TLR signaling, are known effector molecules capable of inhibiting G. duodenalis proliferation.^35,36 However, direct experimental evidence demonstrating that specific activation of the TLR4 or NLRP3 pathways in vivo translates to reduced parasite burden or ameliorated disease pathology in giardiasis is currently lacking. Therefore, future studies employing in vivo infection models combined with genetic (TLR4^−/−, NLRP3^−/− mice) or pharmacological modulation of these pathways are essential to definitively establish their protective roles and evaluate their potential as targets for novel therapeutic or prophylactic strategies against giardiasis.

In conclusion, our study identified GdMIF as a novel immunomodulatory PAMP from G. duodenalis. GdMIF triggered two major signaling axes via TLR4: TLR4-mediated activation of MAPK (p38 and ERK) and AKT pathways, regulating the secretion of IL-6, IL-10, IL-12, TNF-α, and IL-1β. Furthermore, GdMIF-induced NLRP3 inflammasome activation to secrete IL-1β via TLR4 pathway. These findings significantly advanced our understanding of G. duodenalis-host innate immune interactions, demonstrated that GdMIF was a novel PAMP was recognized by TLR4.

Limitations of the study

Although in this study we have identified GdMIF as a novel secreted protein and a PAMP activating TLR4-mediated innate immunity, our primary findings were based on experiments performed with recombinant proteins and mouse peritoneal macrophages in vitro, not fully recapitulate the complexity of the immune response within the native intestinal microenvironment during G. duodenalis infection. Future studies should aim to construct a GdMIF-knockout G. duodenalis strain to accurately compare its immunostimulatory capacity and virulence with wild-type parasites. Additionally, the potential of GdMIF as a vaccine antigen or a detection target warrants further investigation.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Jianhua Li (jianhuali7207@163.com).

Materials availability

The recombinant protein generated in this study is available from the lead contact.

Data and code availability

• •Raw data supporting the findings of this study were deposited on Mendeley at https://data.mendeley.com/datasets/3jjrgv6rgf/1, and the accession number and corresponding dataset link were provided in the key resources table.
• •This study did not generate any original code.
• •No additional resources were generated or analyzed during this study.

Acknowledgments

This study received financial support from the National Natural Science Foundation of China (no. 32202834) and the Jilin Provincial Scientific and Technological Development Program (no. 20220508047RC). Thanks to our colleagues in the laboratory for valuable discussions. We thank Yuanyuan Zhang, technicians from the Instrument Development Center of Jilin University, for the help with the Olympus FluoView FV3000 confocal microscope.

Author contributions

M.C., conceptualization, data curation, investigation, methodology, writing – original draft, writing – review and editing; X.Z., conceptualization, data curation, methodology, writing – original draft, validation; Z.L. and X.W., methodology, supervision, writing – review and editing; X.Z., H.Y., and B.L., methodology; Y.Y. and Z.S., investigation; P.G. and N.Z., supervision; X.L. and J.L., project administration, conceptualization, resources, methodology, supervision, writing – review and editing.

Declaration of interests

The authors declare no competing interests.

STAR★Methods

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Phosphor-p38 (Thr180/Tyr182)	Cell Signaling Technology	Cat#:#9211; RRID:AB_331641
anti-p38	Cell Signaling Technology	Cat#:#9212; RRID:AB_2533100
Phospho-ERK (Thr202/Tyr204)	Cell Signaling Technology	Cat#:#9101; RRID:AB_11009630
ERK	Cell Signaling Technology	Cat#:#4695; RRID:AB_390779
Phospho-AKT (Ser473)	Cell Signaling Technology	Cat#:#15116; RRID:AB_2798713
AKT	Cell Signaling Technology	Cat#:#9272; RRID:AB_329827
Phospho-p65 (Ser536)	Cell Signaling Technology	Cat#:#3033; RRID:AB_331284
NF-κB p65	Cell Signaling Technology	Cat#:#8242; RRID:AB_10859369
Phospho-IκBα (Ser32)	Cell Signaling Technology	Cat#:#9246; RRID:AB_2267145
IκBα	Cell Signaling Technology	Cat#:#4812; RRID:AB_10694416
NLRP3	AdipoGen	Cat#:AG-44B-0009; RRID:AB_3095234
IL-1β	R&D	Cat#:BAF401; RRID:AAB_2139808
caspase-1 (p20)	Adipogen	Cat#:AG-20B–0042B; RRID:AB_2490249
TLR2	HUAAN	Cat#:ET1705-92; RRID:AB_3070621
TLR4	HUAAN	Cat#:RT1666; RRID:AB_3069058
GAPDH	Proteintech	Cat#:60004-1-Ig; RRID:AB_2107436
HRP-conjugated Rabbit Anti-Goat IgG(H + L)	Proteintech	Cat#:SA00001-4; RRID:AB_2864335
HRP-conjugated Goat Anti-Mouse IgG(H + L)	Proteintech	Cat#:SA00001-1; RRID:AB_2722565
HRP-conjugated Goat Anti-Rabbit IgG(H+L)	Proteintech	Cat#:SA00001-2; RRID:AB_2722564
CoraLite488-conjugated Goat Anti-Mouse IgG(H + L)	Proteintech	Cat#:SA00013-1; RRID:AB_2810983
CoraLite594-conjugated Goat Anti-Mouse IgG(H + L)	Proteintech	Cat#:SA00013-3; RRID:AB_2797133
GdMIF	anti-GdMIF mouse serum (Homemade)	–
Bacterial and virus strains
Escherichia coli BL21 (DE3)	TIANGEN	–
Chemicals, peptides, and recombinant proteins
Fetal bovine serum (FBS)	Sigma-Aldrich	Cat#:9014-81-7
Bovine bile	Sigma-Aldrich	Cat#:B3883
Penicillin-Streptomycin solution	Sigma-Aldrich	Cat#:TMS-AB2
Isopropyl-β-D-thiogalactopyranoside (IPTG)	Biosharp	Cat#:BS119-25g
BeyoGold™ GST-tag Purification Resin	Beyotime	Cat#:P2251
Thrombin	Sigma-Aldrich	Cat#:9002-04-4
Triton X-114	Sigma-Aldrich	Cat#:9036-19-5
Difo Fluid Thioglycolate Medium	BD	–
TRIzol	Epizyme	Cat#:15596-026
SYBR Green qPCR Master Mix	Monad	–
p38 inhibitor (SB203580)	Selleck	Cat#:S1076
ERK inhibitor (PD98059)	Selleck	Cat#:S1177
AKT inhibitor (MK-2206 2HCl)	Selleck	Cat#:S1078
RIPA	Beyotime	Cat#:P0013B
Protein-free rapid blocking solution	EpiZyme	Cat#:PS108P
Tris-Buffered Saline with Tween 20 (TBST)	Servicebio	Cat#:G0004
Enhanced chemiluminescence solution	EpiZyme	Cat#:SQ101
LPS	Sigma-Aldrich	Cat#:916374
ATP	Sigma-Aldrich	Cat#:A1852
Critical commercial assays
the Limulus Amebocyte Lysate (LAL) kit	GenScript	Cat#:L00305C
the PrimeScript™ RT reagent Kit	Takara	Cat#:RR037A
Mouse IL-6 Uncoated ELISA Kit	Invitrogen	Cat#:88-7064-88
Mouse IL-10 Uncoated ELISA Kit	Invitrogen	Cat#:88-7105-88
Mouse IL-12/IL-23 (total p40) Uncoated ELISA Kit	Invitrogen	Cat#:88-7120-88
Mouse TNF-α Uncoated ELISA Kit	Invitrogen	Cat#:88-7324-88
Mouse IL-1β Uncoated ELISA Kit	Invitrogen	Cat#:88-7013A-88
LDH cytotoxicity assay kit	Beyotime	Cat#:C0016
DAPI	Beyotime	Cat#:P0131
Deposited data
Raw data of this paper	This paper; Mendeley	Mendeley Data: https://data.mendeley.com/datasets/3jjrgv6rgf/1
Experimental models: Organisms/strains
G. duodenalis trophozoites	ATCC	ATCC 30957
Mouse preparation of mouse peritoneal macrophage (PMϕs)	Primary cell	–
Oligonucleotides
Primer: GdMIF-Forward: 5′-GGAATTCATGCCTTGCGCCATTGTC-3′	This paper	–
Primer: GdMIF-Reverse: 5′-CCTCGAGCTAAAACGTGCTGCCA TTA-3′	This paper	–
Primer for TLR2, see Table S1	This paper	–
Primer for TLR3, see Table S1	This paper	–
Primer for TLR4, see Table S1	This paper	–
Primer for GAPDH, see Table S1	This paper	–
Recombinant DNA
pGEX-4T-1-GdMIF	This paper	–
Software and algorithms
UniProtKB	https://www.uniprot.org	–
AlphaFold	https://alphafold.com/	–
Pymol	https://www.pymol.org/	v3.1.6.1
GraphPad Prism	GraphPad Software LLC	GraphPad Prism 7
ImageJ	NIH	ImageJ 1.53e

Experimental model and study participant details

Mice

Female and male C57BL/6 mice (6–8 weeks old) were purchased from Changsheng Experimental Animal Center (Benxi, China). Female and male TLR2^−/− and TLR4^−/− mice were purchased from the Model Animal Research Center of Nanjing University (Nanjing, China). Female and male NLRP3^−/− mice were obtained from the Jackson Laboratory (Bar Harbor, Maine, USA). All mice were housed under specific pathogen-free conditions with a 12 h light/dark cycle and provided food and water ad libitum. The bred female mice were used for experiments, all animal experiments were approved by the Animal Welfare and Research Ethics Committee of Jilin University (IACUC License No. SY202007001) and conducted in accordance with the relevant guidelines and regulations.

Parasites preparation

G. duodenalis trophozoites (WB strain, ATCC30957) were cultured in modified TYI-S-33 medium with 10% fetal bovine serum (FBS, Sigma-Aldrich, Burlington, USA), 0.1% bovine bile (Sigma-Aldrich, Burlington, USA) and 1% Penicillin-Streptomycin solution (Sigma-Aldrich, Burlington, USA) at 37°C under microaerophilic conditions.

Method details

Expression and purification of rGdMIF

GdMIF gene sequences (GL50803_0012091) and the multiple cloning sites of the pGEX-4T-1 vector (TIANGEN, Beijing, China) were used to design primers. The GdMIF genes were amplified by PCR from G. duodenalis cDNA and linked to the pGEX-4T-1 vector. The constructed expression plasmid (pGEX-4T-1-GdMIF) was identified by enzyme digestion and transferred into Escherichia coli BL21 (DE3) competent cells (TIANGEN, Beijing, China). For rGdMIF expression, the transformed bacteria (pGEX-4T-1-GdMIF) was cultured and induced with 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) (Biosharp, Hefei, China) at 37°C with shaking. Then, the bacterial solution was ultrasonically broken by ultrasonic treatment and centrifuged at 8,000 × g for 5 min to collect the precipitation and supernatant. rGdMIF was purified from the BeyoGold glutathione S-transferase (GST)-tag Purification Resin (Beyotime, Shanghai, China) according to the manufacturer’s instructions.

To remove the GST tag, purified fusion protein was incubated with thrombin (Sigma-Aldrich, MO, USA) at 25 °C for 4 h at a mass ratio of 1:100 (enzyme:substrate). The cleaved mixture was subsequently passed through a glutathione Sepharose column to remove free GST and undigested fusion protein, rGdMIF (tag-free) was obtained.⁴⁹

To remove endotoxin contamination, rGdMIF was treated with 1% Triton X-114 (v/v) (Sigma-Aldrich, MO, USA) at 4°C for 30 min, followed by phase separation at 37°C for 10 min and centrifugation at 12,000 × g for 10 min to collect the aqueous phase. This procedure was repeated three to five times.⁵⁰ Subsequently, residual Triton X-114 was effectively eliminated from the protein solution by extensive dialysis against PBS using a membrane with a molecular weight cutoff of 10 kDa. Endotoxin levels were quantified using the Limulus Amebocyte Lysate (LAL) assay (GenScript, New Jersey, USA), and samples with endotoxin levels below 0.1 EU/mL were used for subsequent experiments.⁵¹

Preparation of mouse peritoneal macrophage (PMϕs)

Mouse was intraperitoneally injected with 2 mL sterile 2.98% Difo Fluid Thioglycolate Medium (BD, Franklin Lakes, USA) and euthanized at 3 days post-injection. The peritoneal cavity was rinsed with 3 mL cold PBS. The collected PMϕs suspension was centrifuged at 1,000 × g for 10 min, and PMϕs were plated in cell culture plates, and cultured in 1640 medium (Sigma-Aldrich, Burlington, USA) with 10% fetal bovine serum (FBS, Sigma-Aldrich, Burlington, USA) and 1% Penicillin-Streptomycin solution (Sigma-Aldrich, Burlington, USA) at 37°C with 5% CO₂ for 6 h.

Detection of secreted GdMIF in G. duodenalis ESP (GdESP)

The G. duodenalis trophozoites were resuspended at a concentration of 1 × 10⁶ parasites/mL in modified TYI-S-33 medium supplemented with depleted fetal bovine serum and maintained under standard culture conditions (37°C, 12 h). Following incubation, cell-free supernatants were obtained through sequential centrifugation steps: initial clarification at 2,000 × g for 10 min (4°C), followed by high-speed centrifugation at 10,000 × g for 45 min (4°C).⁵² The final supernatant fractions were collected and concentrated by the methanol-chloroform method for subsequent analysis. Polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, USA) were used to transfer GdESP protein after SDS-PAGE. The anti-GdMIF mouse serum (1:200) incubated PVDF membrane overnight at 4°C, and then HRP-conjugated Goat Anti-Mouse IgG(H + L) (Proteintech, Wuhan, China) incubated PVDF membrane for 1 h. The membrane was then treated with a chemiluminescent substrate and visualized using the Clinx ChemiScope Series (Clinx, Shanghai, China).

Quantitative real-time PCR (qPCR) detection

WT mouse PMϕs were treated with PBS, rGdMIF (1 μg/mL), and G. duodenalis trophozoites (3 × 10⁶) for 2 h. Mouse PMϕs RNA was isolated using TRIzol reagent (Epizyme, Shanghai, China) and converted to cDNA with the PrimeScript RT reagent Kit (Takara, Dalian, China). The cDNA was amplified using SYBR Green qPCR Master Mix (Monad, Wuhan, China). The qPCR primers used were shown in Table S1. GAPDH was used as an endogenous control. The relative expression of mRNA was calculated by the 2^−ΔΔCq method.

The protein-protein interaction analysis

Proteins 3D structure files were downloaded from UniProtKB (https://www.uniprot.org), and proteins 3D structure were analyzed by the AlphaFold Protein Structure Database (https://alphafold.com/). The final docking result was analyzed by Pymol software (https://www.pymol.org/).

Western blot analysis

WT mouse PMϕs (3 × 10⁶ cells/well) were treated with PBS, rGdMIF at different times (0.5, 1, 2, 4 h, 1 μg/mL) or concentrations (0.01, 0.5, 1, 5, 10, 20 μg/mL, 2 h), and G. duodenalis trophozoites (3 × 10⁶). WT, TLR2^−/− and TLR4^−/− mouse PMϕs were treated with 1 μg/mL rGdMIF for 2 h. For the inhibitor experiment, PMϕs were pretreated with p38 inhibitor (SB203580, 30 μM), ERK inhibitor (PD98059, 40 μM), AKT inhibitor (MK-2206 2HCl, 5 μM) (all from Selleck, Shanghai, China) for 1 h, and then incubated with 1 μg/mL rGdMIF for 2 h. PMϕs were lysed with RIPA (Beyotime, Shanghai, China) and then subjected to low-temperature centrifugation at 12,000 × g for 10 min to collect the protein supernatant. The protein supernatant was collected and mixed with the protein loading buffer and boiled for 10 min to prepare the protein sample. A total of 30 μg of protein per sample was separated using SDS-PAGE and transferred to a PVDF membrane (Millipore, Bedford, USA). Membranes were blocked with a protein-free rapid blocking solution (EpiZyme, Shanghai, China) for 30 min, membranes were incubated with primary antibodies (anti-Phosphor-p38 (Thr180/Tyr182), anti-p38, anti-phospho-ERK (Thr202/Tyr204), anti-ERK, anti-Phospho-AKT (Ser473), anti-AKT, anti-NF-κB p65, anti-Phospho-p65 (Ser536), anti-Phospho-IκBα (Ser32), anti-IκBα (all rabbit), and mouse anti-GAPDH, diluted at 1:1,000 in 5% BSA) (Cell Signaling Technology, Danvers, USA) at 4°C for overnight, washed 3 times with Tris-Buffered Saline with Tween 20 (TBST) (Servicebio, Wuhan, China), incubated with HRP-conjugated Goat Anti-Mouse IgG(H + L) or HRP-conjugated Goat Anti-Rabbit IgG(H + L) (Proteintech, Wuhan, China) at RT for 1 h, then washed with TBST. The membrane was identified using the Clinx ChemiScope Series (Clinx, Shanghai, China) with enhanced chemiluminescence solution from EpiZyme (EpiZyme, Shanghai, China), gray analysis was performed using ImageJ.

WT mouse PMϕs (3 × 10⁶ cells/well) were pretreated with 100 ng/mL LPS (Sigma, MO, USA) for 3 h,^18,53,54 then washed cells for twice, and treated with PBS, rGdMIF (time gradients: 6, 12, 24 h, 1 μg/mL concentration gradients: 0.01, 0.5, 1, 5, 10, 20 μg/mL, 24 h) and G. duodenalis trophozoites (3 × 10⁶), 5 mM ATP (Sigma, MO, USA) for 30 min, respectively. After pretreated with 100 ng/mL LPS for 3 h, WT, NLRP3^−/−, TLR2^−/− and TLR4^−/− mouse PMϕs were treated with 1 μg/mL rGdMIF or 3 × 10⁶ G. duodenalis trophozoites for 24 h. Supernatant and cell lysate were collected. Mouse anti-NLRP3 in dilution 1:2,000 (AdipoGen, Liestal, Switzerland), goat anti-IL-1β in dilution 1:2,000 (R&D Systems, Minneapolis, USA), and mouse anti-caspase-1 (p20) in dilution 1:1,000 (Adipogen, Liestal, Switzerland) were used as the primary antibody. The membranes were incubated with HRP-conjugated Goat Anti-Mouse IgG(H + L) and HRP-conjugated Rabbit Anti-Goat IgG(H + L) (Proteintech, Wuhan, China) at RT for 1 h. The membrane was identified using the Clinx ChemiScope Series (Clinx, Shanghai, China).

Cytokine assays

WT mouse PMϕs (5 × 10⁵ cells/well) were treated with PBS, rGdMIF (0.01, 0.5, 1, 5, 10, 20 μg/mL), and G. duodenalis trophozoites (5 × 10⁵) for 24 h, respectively. For the inhibitor experiment, PMϕs were pretreated with p38 inhibitor (SB203580, 30 μM), ERK inhibitor (PD98059, 40 μM), AKT inhibitor (MK-2206 2HCl, 5 μM) (all from Selleck, Shanghai, China) for 1 h, and then incubated with 1 μg/mL rGdMIF for 24 h. All supernatants were collected for ELISA. ELISA kits were used to measure IL-6, IL-10, TNF-α, IL-12/IL-23 (total p40) and IL-1β in the supernatants (Invitrogen, San Diego, California, USA) according to the manufacturer’s instructions. Absorbance was collected at 450 nm.

Lactate dehydrogenase (LDH) release assay

WT PMϕs (3 × 10⁶ cells/well) were pretreated with 100 ng/mL LPS for 3 h, then treated with PBS, rGdMIF (time gradients: 6, 12, 24 h, 1 μg/mL. Concentration gradients: 0.01, 0.5, 1, 5, 10, 20 μg/mL, 24 h) and 3 × 10⁶ G. duodenalis trophozoites, 5 mM ATP for 30 min, respectively. WT, NLRP3^−/−, TLR2^−/− and TLR4^−/− mouse PMϕs were pretreated with LPS (100 ng/mL) for 3 h, then PMϕs were treated with 1 μg/mL rGdMIF or 3 × 10⁶ G. duodenalis trophozoites for 24 h. The cell culture supernatant was collected and centrifuged at 12000 g for 5 min. LDH release was detected using a commercial LDH cytotoxicity assay kit (Beyotime, Shanghai, China) as the manufacturer’s instructions. Absorbance was recorded at a wavelength of 490 nm.

Immunofluorescence assays (IFA)

WT mouse PMϕs were plated on sterile glass in 24-well plates, pretreated with LPS and treated with PBS, rGdMIF (1 μg/mL), 5 × 10⁵ G. duodenalis trophozoites for 24 h, 5 mM ATP for 30 min. Cells were fixed in 4% paraformaldehyde at RT for 20 min, and permeabilized with 0.5% Triton X-100 at RT for 20 min. Then, cells were blocked in 3% BSA at RT for 1 h and incubated with primary antibodies of NLRP3 (1:200) at 4°C overnight. Next, cells were incubated with the CoraLite594-conjugated Goat Anti-Mouse IgG(H + L) (1:200, Proteintech, Wuhan, China) for 1 h at RT, followed by sealing the slides with 4′,6-diamidino-2-phenylindole (DAPI) (Beyotime, Shanghai, China) containing an anti-fluorescence quench agent. Finally, images were observed using Olympus FV3000 Laser scanning confocal microscope (Olympus, Tokyo, Japan).

Quantification and statistical analysis

Graphs were generated and statistical analyses performed using Prism (GraphPad Software). Data are from three independent experiments (n = 3), and Error bars presented as the mean ± standard deviation (SD). Group comparisons were performed using the unpaired Student’s t test (for two groups) or one-way ANOVA (for multiple groups). n.s. (p > 0.05) indicates not significant, and ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 represent statistically significant difference.

Published: December 11, 2025