Lutein elevates mitochondrial superoxide, suppressing inflammation and shifting pyroptosis to apoptosis

다국어 초록 (Multilingual Abstract)

Inflammation-driven macrophage activation is a central driver of chronic inflammatory diseases, yet the upstream redox mechanisms governing inflammasome priming, pro-inflammatory transcriptional reprogramming, and cell-fate determination remain incompletely understood. Here, we demonstrate that lutein functions as a multi-target anti-inflammatory regulator that modulates mitochondrial redox signaling and suppresses canonical inflammatory pathways in macrophages. Transcriptomic comparison confirmed that J774A.1 cells closely recapitulate BMDM inflammatory responses, validating their use as an immunological model. Lutein pretreatment induced a robust antioxidant shift, characterized by Hmox1/Nrf2 activation and suppression of Nos2, Ptgs2, Cybb, and other ROS/NO- producing enzymes. This redox rebalancing was validated at both mRNA and protein levels.

Mechanistically, lutein broadly repressed NF-κB–NLRP3 inflammasome priming, reduced NLRP3 and pro–IL-1β accumulation, and strongly inhibited caspase-1 activation, IL-1β maturation, and pyroptosis, effectively blocking the entire priming–activation–execution cycle. Lutein simultaneously downregulated NF-κB, ERK, JNK, and p38 core transcriptional modules and mitigated LPS-driven phosphorylation events, indicating multi-layer suppression of inflammatory signaling networks. The anti-inflammatory action was partially dependent on ROS–HO-1, as NAC or ZnPP attenuated lutein-mediated inhibition of COX-2 and pro–caspase-1.

In chronic M1 macrophages, lutein did not reverse established NF-κB–STAT1 inflammatory programs or iNOS, COX-2 expression. However, it consistently increased HO-1, activated autophagy/mitophagy, suppressed Nlrp3, Casp1 transcription, and promoted a shift from pyroptosis toward non-inflammatory apoptosis, evidenced by increased phospho-JNK, cleaved caspase-3, and PARP.

Multi-omics correlation analysis revealed that lutein-induced HO-1 expression and apoptosis activation were strongly associated with mitochondrial ROS rather than total ROS. Functional assays further demonstrated that lutein selectively increased mitochondrial superoxide while exerting minimal effects on cytosolic ROS, indicating that lutein triggers mitochondrial metabolic stress, which in turn elevates mitoROS and subsequently induces HO-1 expression, autophagy, and apoptosis.

Collectively, these findings identify lutein as a mitochondria-centered redox modulator that attenuates acute inflammation by suppressing inflammasome signaling, and in chronic M1 conditions, redirects inflammatory cell death toward apoptosis through HO-1–autophagy–mitoROS networks. Lutein may thus represent a promising therapeutic strategy for controlling both acute and chronic macrophage-driven inflammation.

목차 (Table of Contents)

• Ⅰ. 서론 1
• 1-1. 염증 반응의 생리적 의미와 병태생리적 양면성 1
• 1-2. TLR4 매개 염증 신호전달과 NF-κB 활성화 1
• 1-3. ROS에 의한 NLRP3 inflammasome 활성과 염증성 세포사멸(pyroptosis) 2
• 1-4. 산화 스트레스에 대한 세포 내 방어기전: Nrf2–HO-1 축의 역할 3
• 1-5. 자가포식(Autophagy)에 의한 염증성 세포사멸 억제와 세포사멸 전환 4
• 1-6. 루테인(Lutein)의 항산화·항염증 잠재력과 연구 필요성 5
• Ⅱ. 재료 및 방법 6
• 2-1. J774A.1 Cell Line Culture 6
• 2-2. Cell Viability Assay (CCK-8) 6
• 2-3. Griess Assay for Nitric Oxide Measurement 7
• 2-4. RNA Extraction and cDNA Synthesis 7
• 2-5. Quantitative Real-Time PCR (qRT-PCR) 8
• 2-6. Western Blot Analysis 10
• 2-7. CM-H DCFDA and MitoSOX 11
• 2-8. Annexin V/PI Assay 11
• 2-9. Immunocytochemistry (ICC) 12
• 2-10. RNA-sequencing Processing 13
• 2-11. M1 Macrophage Polarization 13
• 2-12. ELISA for IL-1β Secretion 14
• 2-13. 통계처리 14
• Ⅲ. 결과 16
• 3-1. 마우스 대식 세포주와 1차 배양세포의 면역·대사 반응 비교를 통한 세포 모델의 적합성 검증 16
• 3-2. Lutein 처리에 의한 ROS–HO-1 축 조절 및 항염증성 전사체 변화 22
• 3-3. Lutein 처리에 의한 NF-κB–NLRP3 priming 신호 조절에 의한 canonical inflammasome 활성 억제 가능성 제시 30
• 3-4. Lutein 처리에 의한 NLRP3 inflammasome 활성화 모델에서 priming–activation–pyroptosis 전 과정 억제 검증 36
• 3-5. 전염증성 사이토카인의 하향조절을 통한 M1 대식세포 분극화 억제 효과 44
• 3-6. Lutein 처리에 의한 NF-κB 및 MAPK 경로를 포함한 핵심 염증성 신호전달경로 억제 49
• 3-7. Lutein 처리에 의한 HO-1 의존적 항염증 축과 자가포식 autophagy와 mitopphagy 경로 활성화하여 염증 반응 조절 59
• 3-8. 만성 염증 환경에서 Lutein 처리에 의한 전염증성 M1 대식세포 표현형 변화 분석 65
• 3-9. 만성 염증 환경에서 lutein은 autophagy 활성을 촉진하고 pyroptosis를 apoptosis로 전환한다 71
• 3-10. Mitochondrial metabolic stress의 HO-1 유도 및 세포사멸 촉진 79
• 3-11. Lutein 처리에 의한 미토콘드리아 ROS 선택적 증가 및 HO-1 기반 항염증 프로그램 유도 83
• Ⅳ. 요약 86
• Ⅴ. 참고문헌 88
• Ⅵ. 영문초록 95
• List of Figure
• Figure 1. Differential gene expression profiles in LPS-stimulated J774A.1 macrophages and BMDMs. 17
• Figure 2. Heatmap of commonly regulated inflammation-related genes in J774A.1 cells and BMDM following LPS stimulation. 18
• Figure 3. Functional enrichment analysis of common DEGs in LPS-stimulated J774A.1 and BMDM cells. 20
• Figure 4. CCK-8 analysis of cell viability following lutein treatment. 23
• Figure 5. Transcriptomic analysis reveals antioxidant responses and ROS/NO metabolic reprogramming. 24
• Figure 6. qRT-PCR validation of Hmox1 induction and Nos2/Cox2 suppression. 26
• Figure 7. Lutein suppresses COX-2 and iNOS while inducing HO-1 at the protein level in LPS-stimulated J774A.1 macrophages. 27
• Figure 8. Lutein suppresses inflammasome related gene expression in LPS stimulated J774A.1 macrophages. 31
• Figure 9. Lutein suppresses Nfkbia and Nlrp3 mRNA expression in LPS stimulated J774A.1 macrophages. 32
• Figure 10. Lutein suppresses inflammasome priming markers and modulates autophagy-related proteins at the protein level in LPS stimulated J774A.1 macrophages. 33
• Figure 11. Lutein suppresses NLRP3 inflammasome activation and IL-1β secretion. 37
• Figure 12. Lutein suppresses LPS–Nigericin–induced pyroptotic cell death as assessed by live-cell Annexin V/PI imaging. 39
• Figure 13. Flow cytometric assessment of pyroptosis under LPS–Nigericin stimulation. 41
• Figure 14. Heatmap of pro-inflammatory cytokine gene expression in J774A.1 macrophages treated with lutein and LPS. 43
• Figure 15. Lutein suppresses pro-inflammatory cytokine gene expression. 43
• Figure 16. Lutein suppresses LPS/IFN-γ–induced M1 macrophage polarization in J774A.1 cells. 45
• Figure 17. Transcriptomic distribution of pathway-wise log2 fold change in inflammatory signaling networks. 50
• Figure 18. Time-course western blot analysis of LPS-induced inflammatory signaling in the presence or absence of lutein. 51
• Figure 19. Transcriptomic analysis of the NF-κB core gene panel. 52
• Figure 20. Transcriptomic analysis of the ERK core gene panel. 53
• Figure 21. Transcriptomic analysis of the JNK core gene panel. 54
• Figure 22. Transcriptomic analysis of the p38 MAPK core gene panel. 55
• Figure 23. Anti-inflammatory mechanisms regulated by lutein in combination with an NF-κB inhibitor. 56
• Figure 24. Anti-inflammatory mechanisms regulated by lutein in combination with MAPK inhibitors. 57
• Figure 25. Effects of ROS scavenging and HO-1 inhibition on lutein-mediated anti-inflammatory signaling. 60
• Figure 26. Transcriptomic profiling of ROS-Related and Nrf2-Driven antioxidant gene modulation. 61
• Figure 27. Transcriptomic evidence of autophagy activation following lutein treatment. 62
• Figure 28. Mitophagy activation following lutein treatment. 63
• Figure 29. Effects of post-treatment with lutein on established M1-polarized macrophages. 66
• Figure 30. Transcriptomic enrichment analysis of NF-κB and TLR signaling pathways in M1-polarized macrophages following lutein treatment. 68
• Figure 31. Effects of lutein on Inflammatory protein expression in fully polarized M1 macrophages. 69
• Figure 32. Lutein enhances autophagy programs in M1 macrophages despite established inflammatory polarization. 72
• Figure 33. Transcriptomic heatmap of inflammasome–pyroptosis-related genes in M1 macrophages treated with lutein (RNA-seq). 74
• Figure 34. Transcriptomic GSEA of apoptosis-related pathways in lutein-treated M1 macrophages. 75
• Figure 35. Lutein promotes apoptosis signaling in M1-like macrophages. 77
• Figure 36. Transcriptome-based correlation analysis of antioxidant, mitochondrial, and apoptosis pathways in M1 macrophages treated with lutein. 80
• Figure 37. Lutein selectively elevates mitochondrial superoxide without increasing total cellular ROS. 84
• List of Table
• Table 1. Primers used in qPCR. 9