Calmodulin Tri-methylation by Calmodulin-lysine N-methyltransferase Plays an Anti-inflammatory Role by Inhibiting caspase-11 Noncanonical Inflammasome in Macrophages

다국어 초록 (Multilingual Abstract)

The caspase-11 noncanonical inflammasome is activated in response to lipopolysaccharide (LPS), a highly immunogenic endotoxin present in the outer membrane of Gram-negative bacteria. and is closely associated with the pathogenesis of sepsis. Activation of caspase-11 induces gasdermin D (GSDMD) cleavage, pyroptosis, and the release of IL-1β and IL-18, collectively driving severe inflammatory responses such as acute lethal sepsis. However, the intrinsic cellular mechanisms that negatively regulate this pathway remain poorly understood.
In this study, we investigated whether Lys116 tri-methylation of calmodulin (CaM)—a major calcium-binding regulatory protein—modulates caspase-11 noncanonical inflammasome responses. We found that expression of calmodulin-lysine N-methyltransferase (CAMKMT) was markedly reduced during caspase-11 activation, leading to decreased CaM Lys116 tri-methylation and suppression of CaM-dependent signaling, including the CaMKK2–CaMKIV phosphorylation axis. Overexpression of CAMKMT restored CaM trimethylation, normalized CaM-dependent phosphorylation signaling, and significantly inhibited caspase-11 and GSDMD cleavage, pyroptosis, and IL-1β and IL-18 production.
Furthermore, introduction of a trimethylation-defective CaM mutant (K116A), in which Lys116 was substituted with alanine, abolished the anti-inflammatory effects, demonstrating that CaM Lys116 tri-methylation is essential for caspase-11 noncanonical inflammasome regulation. In an in vivo acute lethal sepsis model,
CaM WT significantly improved survival, reduced sepsis scores, and attenuated systemic inflammation, whereas CaM K116A further increased inflammatory responses
Collectively, our findings identify CaM Lys116 tri-methylation as a critical regulator of caspase-11 noncanonical inflammasome activation and highlight the CaM Lys116 methylation as a potential therapeutic target for acute inflammatory diseases.

국문 초록 (Abstract)

Caspase-11 비고전적 염증복합체는 그람 음성균의 세포막에서 유래한 강한 내독소인 지질다당류(LPS)에 의해 유도되며, 이는 급성 패혈증의 발병기전과 밀접하게 연관되어 있다. 활성화된 Caspase-11 비고전적 염증복합체는 GSDMD 절단, 염증성 세포사, 그리고 IL-1β 및 IL-18의 분비를 유도하여 급성 패혈증과 같은 전신 염증반응을 일으킨다. 그러나 이 경로를 조절하는 세포 내 기전은 아직 명확히 규명되지 않았다.
본 연구에서는 주요 칼슘 결합 조절 단백질인 칼모듈린의 라이신116 삼중메틸화라는 번역 후 변형(PTM)이 Caspase-11 비고전적 인플라마좀 활성에 미치는 영향을 조사하였다. 연구 결과, 칼모듈린 메틸화효소인 CAMKMT의 발현은 Caspase-11 비고전적 염증복합체 활성화 동안 현저히 감소하였으며, 이는 칼모듈린 라이신116 삼중메틸화의 감소 및 CaMKK2, CaMKIV 인산화 억제로 이어졌다. 칼모듈린 메틸화효소를 과발현시키면 칼모듈린 삼중메틸화가 회복되고, 칼모듈린 의존적 인산화 신호가 정상화되었으며, Caspase-11과 GSDMD 절단, 염증성 세포사, IL-1β 및 IL-18 생성이 유의하게 억제되었다.
더 나아가, 라이신116이 알라닌으로 치환된 삼중메틸화 결함 칼모듈린 돌연변이(K116A)를 도입하였을 때 염증성 세포사가 증가하였으며, 이는 칼모듈린 라이신 116 삼중메틸화가 Caspase-11 비고전적 염증복합체 활성 조절에 필수적임을 보여주었다. 생체 내 급성 패혈증 모델에서도 칼모듈린 WT형은 생존율 증가, 패혈증 점수 감소, 전신 염증 완화 효과를 보였던 반면, 칼모듈린 K116A 변이형는 오히려 염증 반응을 증가시켰다.
종합적으로, 본 연구는 칼모듈린 라이신116 삼중메틸화가 Caspase-11 비고전적 염증복합체 활성화의 중요한 조절자임을 규명하였으며, 칼모듈린 라이신116 메틸화가 급성 염증성 질환의 잠재적 치료 표적이 될 수 있음을 제시한다.

목차 (Table of Contents)

• Chapter Ⅰ. Calmodulin Tri-methylation by Calmodulin-lysine N-methyltransferase Plays an Anti-inflammatory Role by Inhibiting caspase-11 Noncanonical Inflammasome in Macrophages 1
• 1. Introduction 2
• 2. Materials and Methods 5
• 2.1. Cell culture 5
• 2.2. Plasmid construction 5
• 2.3. Lentiviral production and transduction 6
• 2.4. Pyroptosis assay 6
• 2.5. Immunoblotting 7
• 2.6. Immunoprecipitation 7
• 2.7. Cytokine measurement 8
• 2.8. RNA extraction and PCR 8
• 2.9. LPS-induced acute lethal sepsis model 9
• 2.10. Statistical analysis 12
• 3. Results 13
• 3.1. CAMKMT was downregulated during caspase-11 noncanonical inflammasome-activated inflammatory responses in macrophages 13
• 3.2. Tme-K & function of CaM were inhibited during caspase-11 noncanonical inflammasome-activated inflammatory responses in macrophages 14
• 3.3. CAMKMT induced Tme-K & function of CaM during caspase-11 noncanonical inflammasome-activated inflammatory responses in macrophages 16
• 3.4. CAMKMT inhibited caspase-11 noncanonical inflammasome-activated inflammatory responses in macrophages 18
• 3.5. CAMKMT-mediated CaM Tme-K inhibited caspase-11 noncanonical inflammasome-activated inflammatory responses in macrophages 20
• 3.6. CaM Tme-K mitigated acute lethal sepsis in mice 22
• 4. Discussion 23
• 5. Conclusion 25
• Chapter 2. Silymarin in Silybum marianum Ameliorated Acute Lethal Sepsis in Mice by Targeting Caspase-11 Noncanonical Inflammasome in Macrophages 50
• 1. Introduction 51
• 2. Materials and Methods 53
• 2.1. Materials 53
• 2.2. Acute lethal sepsis in mice 54
• 2.3. Cell culture 57
• 2.4. Cell viability assay 57
• 2.5. Pyroptosis assay 58
• 2.6. Flow cytometry analysis 58
• 2.7. Enzyme-linked immunosorbent assay 59
• 2.8. Nitric oxide (NO) assay 59
• 2.9. Quantitative real-time PCR (qPCR) 60
• 2.10. Western blot 61
• 2.11. Caspase-11-LPS binding assay 61
• 2.12. Statistical analysis 62
• 3. Results 63
• 3.1. Silymarin alleviated acute lethal sepsis in mice 63
• 3.2. Silymarin reduced caspase-11 inflammasome-mediated pyroptosis in macrophages 64
• 3.3. Silymarin suppressed caspase-11 inflammasome– dependent production of inflammatory mediators 65
• 3.4. Mechanisms of silymarin-mediated inhibition of caspase-11 inflammasome activation 66
• 4. Discussion 67
• 5. Conclusion 70
• Chapter 3. NIBR1453 Inhibits Caspase-11 Noncanonical Inflammasome Activation in Macrophages 86
• 1. Introduction 87
• 2. Materials and Methods 88
• 2.1. Cell culture 88
• 2.2. Reagents and stimulation 88
• 2.3. Cytotoxicity assay 88
• 2.4. Pyroptosis assay 89
• 2.5. Enzyme-linked immunosorbent assay 89
• 2.6. RNA extraction and quantitative PCR 90
• 2.7. Immunoblotting 90
• 2.8. Statistical analysis 90
• 3. Results 91
• 3.1. Evaluation of cytotoxicity and determination of non-toxic concentrations of NIBR1453. 91
• 3.2. NIBR1453 suppresses caspase-11–dependent pyroptosis more effectively than diclofenac. 92
• 3.3. NIBR1453 reduces caspase-11–dependent cytokine production. 93
• 3.4. NIBR1453 suppresses mRNA expression of caspase-11 –related inflammatory genes 94
• 3.5. NIBR1453 decreases protein levels associated with caspase-11 activation 95
• 4. Discussion 96
• 5. Conclusion 98
• References 107
• Abstract in Korean(국문요지) 120