Knockdown of ITIH4 reduces inflammatory damage and apoptosis of A549 cells induced by Mycoplasma pneumoniae through NLRP3 inflammation
Main Article Content
Keywords
apoptosis, inflammation, ITIH4, Mycoplasma pneumonia, NLRP3
Abstract
Background: Mycoplasma pneumoniae (MP) is a leading cause of community-acquired respiratory infections in pediatric patients. This study aimed to investigate whether the pro-inflammatory function of inter-alpha-trypsin inhibitor heavy chain (ITIH4) contributes to the pathogenesis of MP-induced pneumonia.
Method: A549 cells were stimulated with MP to model pneumonia in vitro. ITIH4 expression was knocked down in A549 cells using lentiviral transfection. Cell viability was measured using the Cell Counting Kit-8 (CCK-8) assay, while cell apoptosis was assessed via flow cytometry. The concentrations of pro-inflammatory (IL-6, TNF-α) and anti-inflammatory (IL-10) cytokines were quantified using enzyme-linked immunosorbent assay (ELISA). Western blotting was conducted to detect apoptosis-related proteins and components of the NLRP3 inflammasome.
Result: MP stimulation led to increased ITIH4 expression in A549 cells, and knockdown of ITIH4 prevented the MP-induced reduction in cell viability. Moreover, ITIH4 knockdown reduced the release of inflammatory cytokines in response to MP and significantly decreased MP-induced apoptosis. In addition, ITIH4 knockdown inhibited activation of the NLRP3 inflammasome, while reactivation of NLRP3 reversed the protective effects associated with ITIH4 knockdown.
Conclusion: ITIH4 knockdown alleviates MP-induced inflammatory damage and cell death in A549 cells by inhibiting activation of the NLRP3 inflammasome.
References
2 Ding G, Zhang X, Vinturache A, van Rossum AMC, Yin Y, Zhang Y. Challenges in the treatment of pediatric Mycoplasma pneumoniae pneumonia. Eur J Pediatr. 2024;183(7):3001-11. 10.1007/s00431-024-05519-1
3 Kumar S, Kumar S. Mycoplasma pneumoniae: Among the smallest bacterial pathogens with great clinical significance in children. Indian J Med Microbiol. 2023;46:100480. 10.1016/j.ijmmb.2023.100480
4 Esposito S, Argentiero A, Gramegna A, Principi N. Mycoplasma pneumoniae: a pathogen with unsolved therapeutic problems. Expert Opin Pharmacother. 2021;22(9):1193-202. 10.1080/14656566.2021.1882420
5 He JE, Qu H, Gao CY. Association between inflammation factors and Mycoplasma pneumoniae in children: Protocol for a systematic review. Medicine (Baltimore). 2019;98(15):e15118. 10.1097/MD.0000000000015118
6 Cheng J, Ji D, Yin Y, Wang S, Song K, Pan Q, et al. Proteomic profiling of serum small extracellular vesicles reveals immune signatures of children with pneumonia. Transl Pediatr. 2022;11(6):891-908. 10.21037/tp-22-134
7 Pihl R, Jensen RK, Poulsen EC, Jensen L, Hansen AG, Thogersen IB, et al. ITIH4 acts as a protease inhibitor by a novel inhibitory mechanism. Sci Adv. 2021;7(2). 10.1126/sciadv.aba7381
8 Ma Y, Li R, Wang J, Jiang W, Yuan X, Cui J, et al. ITIH4, as an inflammation biomarker, mainly increases in bacterial bloodstream infection. Cytokine. 2021;138:155377. 10.1016/j.cyto.2020.155377
9 Wen N, Zhao N, Xu H, Zhao Y, Ma J. Serum inter-alpha-trypsin inhibitor heavy chain 4 in patients with inflammatory bowel disease: correlation with disease risk, inflammation, activity, and its variation after treatment. Ir J Med Sci. 2022;191(5):2105-11. 10.1007/s11845-021-02837-3
10 Zhao X, Guo Y, Li L, Li Y. Longitudinal change of serum inter-alpha-trypsin inhibitor heavy chain H4, and its correlation with inflammation, multiorgan injury, and death risk in sepsis. J Clin Lab Anal. 2023;37(3):e24834. 10.1002/jcla.24834
11 Liu F, Liu T, Sun M, Zhou J, Xue F, Chen S, et al. Maxing Shigan Decoction Mitigates Mycoplasma pneumonia-Induced Pyroptosis in A549 Cells via the NLRP3 Inflammasome. Infect Drug Resist. 2021;14:859-67. 10.2147/IDR.S292413
12 Chi Y, Di Q, Han G, Li M, Sun B. Mir-29b mediates the regulation of Nrf2 on airway epithelial remodeling and Th1/Th2 differentiation in COPD rats. Saudi J Biol Sci. 2019;26(8):1915-21. 10.1016/j.sjbs.2019.07.011
13 Luo H, He J, Qin L, Chen Y, Chen L, Li R, et al. Mycoplasma pneumoniae lipids license TLR-4 for activation of NLRP3 inflammasome and autophagy to evoke a proinflammatory response. Clin Exp Immunol. 2021;203(1):66-79. 10.1111/cei.13510
14 Li QL, Wu YY, Sun HM, Gu WJ, Zhang XX, Wang MJ, et al. The role of miR-29c/B7-H3/Th17 axis in children with Mycoplasma pneumoniae pneumonia. Ital J Pediatr. 2019;45(1):61. 10.1186/s13052-019-0655-5
15 Li W, Wang X, An H. Linkage of serum ITIH4 with Th2 signature cytokine, inflammation, exacerbation risk and severity in childhood asthma. Biomark Med. 2024;18(13-14):593-602. 10.1080/17520363.2024.2366149
16 Ding Y, Chu C, Li Y, Li G, Lei X, Zhou W, et al. High expression of HMGB1 in children with refractory Mycoplasma pneumoniae pneumonia. BMC Infect Dis. 2018;18(1):439. 10.1186/s12879-018-3346-8
17 Li G, Fan L, Wang Y, Huang L, Wang M, Zhu C, et al. High co-expression of TNF-alpha and CARDS toxin is a good predictor for refractory Mycoplasma pneumoniae pneumonia. Mol Med. 2019;25(1):38. 10.1186/s10020-019-0105-2
18 Saraiva M, Vieira P, O’Garra A. Biology and therapeutic potential of interleukin-10. J Exp Med. 2020;217(1). 10.1084/jem.20190418
19 Li L, Zhang Y, Zhao L, Shi Y. C-reactive protein-induced injury in Mycoplasma pneumoniae-infected lung epithelial cells is mediated by the P38 MAPK/mitochondrial apoptosis pathway. Microbiol Spectr. 2025;13(3):e0162624. 10.1128/spectrum.01626-24
20 Chen XY, Feng PH, Han CL, Jheng YT, Wu CD, Chou HC, et al. Alveolar epithelial inter-alpha-trypsin inhibitor heavy chain 4 deficiency associated with senescence-regulated apoptosis by air pollution. Environ Pollut. 2021;278:116863. 10.1016/j.envpol.2021.116863
21 Fu J, Wu H. Structural Mechanisms of NLRP3 Inflammasome Assembly and Activation. Annu Rev Immunol. 2023;41:301-16. 10.1146/annurev-immunol-081022-021207
22 Chen G, Wang Y, Zhang Y, Gu F, Yu T. Ursolic acid inhibits NLRP3 inflammasome activation and alleviates vascular smooth muscle injury in Kawasaki disease. Signa Vitae. 2024;20(4).
23 Segovia JA, Chang TH, Winter VT, Coalson JJ, Cagle MP, Pandranki L, et al. NLRP3 Is a Critical Regulator of Inflammation and Innate Immune Cell Response during Mycoplasma pneumoniae Infection. Infect Immun. 2018;86(1). 10.1128/IAI.00548-17
24 Poddighe D, Comi EV, Brambilla I, Licari A, Bruni P, Marseglia GL. Increased Total Serum Immunoglobulin E in Children Developing Mycoplasma pneumoniae-related Extra-pulmonary Diseases. Iran J Allergy Asthma Immunol. 2018;17(5):490-6
25 Zhou L, Li Y, Xu Z, Peng X, Gong X, Yang L. Increased Total Serum Immunoglobulin E Is Likely to Cause Complications of Mycoplasma pneumoniae Pneumonia in Children. Front Cell Infect Microbiol. 2021;11:783635. 10.3389/fcimb.2021.783635
