Arachidonic acid activates NLRP3 inflammasome in MDSCs via FATP2 to promote post-transplant tumour recurrence in steatotic liver grafts

Abstract

Background & aims: The steatotic grafts have been applied in liver transplantation frequently owing to the high incidence of non-alcoholic fatty liver disease. However, fatty livers are vulnerable to graft injury. Myeloid-derived suppressor cell (MDSC) recruitment during liver graft injury promotes tumour recurrence. Lipid metabolism exerts the immunological influence on MDSCs in tumour progression. Here, we aimed to explore the role and mechanism of inflammasome activation in MDSCs induced by lipid metabolism during fatty liver graft injury and the subsequent effects on tumour recurrence.

Methods: MDSC populations and nucleotide-binding oligomerisation domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome levels were investigated in a clinical cohort and a rat liver transplantation model. The mechanism of NLRP3 activation by specific fatty acids was explored in mouse hepatic ischaemia/reperfusion injury (IRI) with tumour recurrence model and in vitro studies.

Results: MDSC populations and NLRP3 levels were increased with higher tumour recurrent rate in patients using steatotic grafts. NLRP3 was upregulated in MDSCs with lipid accumulation post mouse fatty liver IRI. Mechanistically, arachidonic acid was discovered to activate NLRP3 inflammasome in MDSCs through fatty acid transport protein 2 (FATP2), which was identified by screening lipid uptake receptors. The mitochondrial dysfunction with enhanced reactive oxygen species bridged arachidonic acid uptake and NLRP3 activation in MDSCs, which subsequently stimulated CD4⁺ T cells producing more IL-17 in fatty liver IRI. Blockade of FATP2 inhibited NLRP3 activation in MDSCs, IL-17 production in CD4⁺ T cells, and the tumour recurrence post fatty liver IRI.

Conclusions: During fatty liver graft injury, arachidonic acid activated NLRP3 inflammasome in MDSCs through FATP2, which subsequently stimulated CD4⁺ T cells producing IL-17 to promote tumour recurrence post transplantation.

Impact and implications: The high incidence of non-alcoholic fatty liver disease resulted in the frequent application of steatotic donors in liver transplantation. Our data showed that the patients who underwent liver transplantation using fatty grafts experienced higher tumour recurrence. We found that arachidonic acid activated NLRP3 inflammasome in MDSCs through FATP2 during fatty liver graft injury, which led to more IL-17 secretion of CD4⁺ T cells and promoted tumour recurrence post transplantation. The inflammasome activation by aberrant fatty acid metabolism in MDSCs bridged the acute-phase fatty liver graft injury and liver tumour recurrence.

Keywords: Inflammasome; Lipid metabolism; MDSC; Steatotic liver graft; Tumour recurrence.

Graphical abstract

Fig. 1

MDSC populations and NLRP3 levels were increased in patients with HCC who received fatty grafts post liver transplantation. (A) Circulatory and intragraft MDSCs were increased in patients who received fatty grafts compared with those with normal liver grafts (n = 45). Scale bars: 50 μm. (B) The mRNA expressions of NLRP3 and IL-1β were higher in fatty liver grafts (n = 31). (C) More infiltrated NLRP3- and FATP2-positive cells were found in patients who received fatty liver grafts (n = 23). Scale bars: 50 μm. (D) CD33 (a MDSC marker) was colocalised with FATP2 and NLRP3 (n = 23). Scale bars: 5 μm. Error bars indicate SEM; ∗p <0.05, ∗∗p <0.01. FATP2, fatty acid transport protein 2; HCC, hepatocellular carcinoma; MDSC, myeloid-derived suppressor cell; NLRP3, nucleotide-binding oligomerisation domain-like receptor family pyrin domain containing 3; PBMC, peripheral blood mononuclear cell; SSC: side scatter.

Fig. 2

Higher MDSCs and NLRP3 were accompanied with aberrant lipid metabolism and tumour-favouring alteration in fatty grafts post rat liver transplantation. (A) More MDSCs were found in rats implanted with fatty liver grafts. Scale bars: 50 μm. (B) The mRNA expressions of NLRP3 and IL-1β were increased in fatty liver grafts. (C) NLRP3- and FATP2-positive cells were more infiltrated in steatotic grafts and colocalised with CD11b/c (an MDSC marker). Scale bars: 50 μm (left) and 5 μm (right). (D) The lipid metabolism (including arachidonic acid metabolism) was aberrant in fatty grafts through KEGG analysis of RNA-seq post transplantation. (E) Cholesterol homoeostasis, fatty acid metabolism, epithelial mesenchymal transition, and angiogenesis were significantly changed in steatotic grafts by GSEA. (A–C) n = 4/group; (D and E) n = 3/group. Error bars indicate SEM; ∗p <0.05, ∗∗p <0.01, ∗∗∗p <0.001. FATP2, fatty acid transport protein 2; GSEA, gene set enrichment analysis; KEGG, Kyoto Encyclopedia of Genes and Genomes; MDSC, myeloid-derived suppressor cell; NLRP3, nucleotide-binding oligomerisation domain-like receptor family pyrin domain containing 3; PBMC, peripheral blood mononuclear cell; RNA-seq, RNA sequencing.

Fig. 3

Accumulated lipids enhanced NLRP3 in MDSCs of mouse fatty liver post IRI. (A) More M-MDSCs were accumulated in mouse fatty liver after IRI. (B) NLRP3 was upregulated in liver and splenic MDSCs post fatty liver IRI. (C) More FL C16 was absorbed by MDSCs in fatty liver. (D) The neutral lipids (493/503) were accumulated in MDSCs of fatty liver post IRI. (A–D) n = 5/group. Error bars indicate SEM; ∗p <0.05, ∗∗p <0.01. G-MDSC, granulocytic MDSC; IRI, ischaemia/reperfusion injury; M-MDSC, monocytic MDSC; MDSC, myeloid-derived suppressor cell; MFI, mean fluorescence intensity; NLRP3, nucleotide-binding oligomerisation domain-like receptor family pyrin domain containing 3; T-MDSC, total MDSC.

Fig. 4

Arachidonic acid activated NLRP3 inflammasome in MDSCs through FATP2. (A) C18:0 and C20:4 n6 (AA) were screened out in rat fatty vs. normal liver graft after getting rid of the fatty acids as a result of the diet by GC-MS (n = 4/group). (B) FATP2 and CD36 mRNA levels were upregulated in both liver and splenic MDSCs post mouse fatty liver IRI (n = 5/group). (C) FL C16 was augmented by AA stimulation but inhibited by Lipo (an FATP2 inhibitor) in primary MDSCs. (D) AA increased NLRP3, which was reduced by Lipo in primary MDSCs. (E) FATP2 was accumulated in MDSCs by AA stimulation but decreased by Lipo. (F) FATP2 and NLRP3 activation pathway was enhanced by AA and inhibited by FATP2 blockade through Western blot analysis. Scale bars: 10 μm. Error bars indicate SEM; ∗p <0.05, ∗∗p <0.01, ∗∗∗p <0.001. AA, arachidonic acid; ASC, apoptosis-associated speck-like protein containing a caspase recruitment domain; FATP2, fatty acid transport protein 2; FMO, fluorescence minus one; G-MDSC, granulocytic MDSC; GC-MS, gas chromatography–mass spectrometry; IRI, ischaemia/reperfusion injury; Lipo, lipofermata; M-MDSC, monocytic MDSC; MDSC, myeloid-derived suppressor cell; MFI, mean fluorescence intensity; NC, negative control (the primary MDSCs without stimulation); NLRP3, nucleotide-binding oligomerisation domain-like receptor family pyrin domain containing 3; T-MDSC, total MDSC.

Fig. 5

NLRP3 inflammasome activation with mitochondrial dysfunction in MDSCs stimulated naive CD4⁺ T cells producing IL-17. (A) AA increased the ROS (MitoSOX) of primary MDSCs, which was diminished by Lipo through flow cytometry analysis. (B) The MitoSOX was raised by AA and reduced by FATP2 inhibition by immunostaining. Scale bars: 2.5 μm. (C) DHR was enhanced and decreased by AA and Lipo, respectively. Scale bars: 5 μm. (D) The production of IL-17 in primary naive CD4⁺ T cells was increased, decreased, and restored after coculture with MDSCs treated by AA, Lipo, and IL-1β recombinant protein, respectively. (E) FATP2 in hepatic and splenic MDSCs was upregulated after fatty liver IRI in mice. (F) The ROS in MDSCs of the fatty liver was enhanced post IRI in mice. (G) The population of IL-17⁺ CD4⁺ T cells was accumulated after fatty liver IRI. (E–G) n = 5/group. Error bars indicate SEM; ∗p <0.05, ∗∗p <0.01, ∗∗∗p <0.001. AA, arachidonic acid; DHR, dihydrorhodamine 123; FATP2, fatty acid transport protein 2; FMO, fluorescence minus one; G-MDSC, granulocytic MDSC; IRI, ischaemia/reperfusion injury; Lipo, lipofermata; M-MDSC, monocytic MDSC; MDSC, myeloid-derived suppressor cell; MFI, mean fluorescence intensity; NC, negative control (the primary MDSCs without stimulation); NLRP3, nucleotide-binding oligomerisation domain-like receptor family pyrin domain containing 3; ROS, reactive oxygen species; T-MDSC, total MDSC.

Fig. 6

FATP2 blockade diminished NLRP3 activation in MDSCs and IL-17 production in CD4⁺ T cells after mouse fatty liver IRI. (A) The counts of hepatic MDSCs were reduced by FATP2 blockade after fatty liver IRI in mice. (B) NLRP3 in M-MDSCs of the fatty liver was decreased by FATP2 inhibition. (C) Lipo (an FATP2 inhibitor) reduced the FATP2 levels of M-MDSCs post fatty liver IRI. (D) ROS in M-MDSCs was diminished by FATP2 blockade. (E) The population of liver IL-17⁺CD4⁺ T cells was decreased by FATP2 inhibition. (A–E) n = 5/group. Error bars indicate SEM; ∗p <0.05. FATP2, fatty acid transport protein 2; G-MDSC, granulocytic MDSC; IRI, ischaemia/reperfusion injury; Lipo, lipofermata; M-MDSC, monocytic MDSC; MDSC, myeloid-derived suppressor cell; MFI, mean fluorescence intensity; NLRP3, nucleotide-binding oligomerisation domain-like receptor family pyrin domain containing 3; ROS, reactive oxygen species; T-MDSC, total MDSC.

Fig. 7

Targeting FATP2 inhibited liver tumour recurrence in mice. (A) The tumour size was increased in the fatty liver, and decreased and restored by Lipo and IL-1β recombinant protein injection, respectively. (B) FATP2 blockade inhibited the increased tumour size in the fatty liver, whereas IL-1β injection offset the inhibition effects in the mouse tumour recurrence model. (C) IL-17⁺ CD4⁺ T cells were accumulated in the fatty liver, and reduced and raised by Lipo and IL-1β, respectively. (D) The population of liver M-MDSCs was increased in the fatty liver and decreased by FATP2 blockade. (E) NLRP3 in liver MDSCs was upregulated in the fatty liver and reduced by FATP2 inhibition. (F) FATP2 in hepatic MDSCs was enhanced in the fatty liver, whereas Lipo inhibited its levels in M-MDSCs. (G) The ROS in liver M-MDSCs was increased in the fatty liver and diminished by FATP2 inhibition. (A–G) n = 5/group. Error bars indicate SEM; ∗p <0.05, ∗∗p <0.01, ∗∗∗p <0.001. FATP2, fatty acid transport protein 2; FMO, fluorescence minus one; G-MDSC, granulocytic MDSC; Lipo, lipofermata; M-MDSC, monocytic MDSC; MDSC, myeloid-derived suppressor cell; MFI, mean fluorescence intensity; NLRP3, nucleotide-binding oligomerisation domain-like receptor family pyrin domain containing 3; ROS, reactive oxygen species; T-MDSC, total MDSC.