Neutrophil Extracellular Trap-Related Genes and Immune Infiltration in Diabetic Foot Ulcers by Bioinformatics Analysis and Experimental Verification
Journal: Journal of Clinical Medicine Research DOI: 10.32629/jcmr.v5i1.1784
Abstract
The study aims to investigate the correlation between diabetic foot ulcer (DFU) and neutrophil extracellular traps (NETs), as well as to identify potential biomarkers. Gene microarray data from the Gene Expression Omnibus (GEO) was employed in this research. The GSE134431 and GSE80178 datasets were obtained for bioinformatics analysis to identify differentially expressed genes, which were then cross-referenced with a curated database of genes associated with neutrophil extracellular traps (NETs). Next, the specificity of these potential genes for the disease was determined via Receiver Operating Characteristic (ROC) analysis. Subsequently, the protein-protein interaction (PPI) networks and the Least Absolute Shrinkage and Selection Operator (Lasso) regression were used to identify potential key genes. Additionally, the MCPcounter algorithm was utilized to evaluate the immune infiltration and analyze the relationship between core genes and immune cell infiltration. Furthermore, the levels of expression of these candidate genes were verified by RT-qPCR. Four core genes (CXCL8, S100A12, CXCL12, S100A9) were identified through ROC and Lasso regression analyses. Moreover, individuals suffering from diabetic foot disease exhibited decreased expression levels of T cells, CD8 T cells, cytotoxic lymphocytes, NK cells, fibroblasts, and myeloid dendritic cells; In contrast, monocytes and neutrophils exhibited elevated expression levels. The expression levels of CXCL12 and CXCL8 were positively linked to a wide range of immune and endothelial cells, whereas S100A12 and S100A9 showed distinct correlation patterns with specific immune cell types. qPCR analysis showed increased expression of CXCL8 and CXCL12 as the levels of glucose increased in vitro experimental hyperglycemia. In conclusion, the intricate interplay among genes and immune cell types suggests that DFU and NETs hold potential as valuable biomarkers for the diagnosis and treatment of these conditions, thereby enhancing their clinical meanings.
Keywords
diabetic foot ulcer, neutrophil extracellular traps, bioinformatics, immune infiltration, lasso regression
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[31]Lou M, Luo P, Tang R, Peng Y, Yu S, Huang W, et al. Relationship between neutrophil-lymphocyte ratio and insulin resistance in newly diagnosed type 2 diabetes mellitus patients. BMC Endocr Disord. 2015;15:9.
[32]Shiny A, Bibin YS, Shanthirani CS, Regin BS, Anjana RM, Balasubramanyam M, et al. Association of neutrophil-lymphocyte ratio with glucose intolerance: an indicator of systemic inflammation in patients with type 2 diabetes. Diabetes Technol Ther. 2014;16(8):524-30.
[2]Jeyaraman K, Berhane T, Hamilton M, Chandra AP, Falhammar H. Mortality in patients with diabetic foot ulcer: a retrospective study of 513 cases from a single Centre in the Northern Territory of Australia. BMC Endocr Disord. 2019;19(1):1.
[3]Lim JZ, Ng NS, Thomas C. Prevention and treatment of diabetic foot ulcers. J R Soc Med. 2017;110(3):104-9.
[4]Rawat K, Shrivastava A. Neutrophils as emerging protagonists and targets in chronic inflammatory diseases. Inflamm Res. 2022;71(12):1477-88.
[5]Brinkmann V, Reichard U, Goosmann C, Fauler B, Uhlemann Y, Weiss DS, et al. Neutrophil extracellular traps kill bacteria. Science. 2004;303(5663):1532-5.
[6]Lee YS, Kang SU, Lee MH, Kim HJ, Han CH, Won HR, et al. GnRH impairs diabetic wound healing through enhanced NETosis. Cell Mol Immunol. 2020;17(8):856-64.
[7]Wong SL, Demers M, Martinod K, Gallant M, Wang Y, Goldfine AB, et al. Diabetes primes neutrophils to undergo NETosis, which impairs wound healing. Nat Med. 2015;21(7):815-9.
[8]Davis S, Meltzer PS. GEOquery: a bridge between the Gene Expression Omnibus (GEO) and BioConductor. Bioinformatics. 2007;23(14):1846-7.
[9]Sawaya AP, Stone RC, Brooks SR, Pastar I, Jozic I, Hasneen K, et al. Deregulated immune cell recruitment orchestrated by FOXM1 impairs human diabetic wound healing. Nature communications. 2020;11(1):4678.
[10]Ramirez HA, Pastar I, Jozic I, Stojadinovic O, Stone RC, Ojeh N, et al. Staphylococcus aureus Triggers Induction of miR-15B-5P to Diminish DNA Repair and Deregulate Inflammatory Response in Diabetic Foot Ulcers. J Invest Dermatol. 2018;138(5):1187-96.
[11]Schadow. Bioinformatics and Computational Biology Solutions Using R and Bioconductor.Edited by Robert Gentleman, Wolfgang Huber, Vincent J. Carey, Rafael A. Irizarry and Sandrine Dudoit. Brief Bioinform. 2006;8(2):136-7.
[12]Yu G, Wang LG, Han Y, He QY. clusterProfiler: an R package for comparing biological themes among gene clusters. OMICS. 2012;16(5):284-7.
[13]13. Becht E, Giraldo NA, Lacroix L, Buttard B, Elarouci N, Petitprez F, et al. Estimating the population abundance of tissue-infiltrating immune and stromal cell populations using gene expression. Genome Biol. 2016;17(1):218.
[14]Zhu Y, Xia X, He Q, Xiao QA, Wang D, Huang M, et al. Diabetes-associated neutrophil NETosis: pathogenesis and interventional target of diabetic complications. Front Endocrinol (Lausanne). 2023;14:1202463.
[15]Huang Y, Ding Y, Wang B, Ji Q, Peng C, Tan Q. Neutrophils extracellular traps and ferroptosis in diabetic wounds. Int Wound J. 2023;20(9):3840-54.
[16]Shafqat A, Abdul Rab S, Ammar O, Al Salameh S, Alkhudairi A, Kashir J, et al. Emerging role of neutrophil extracellular traps in the complications of diabetes mellitus. Frontiers in medicine. 2022;9:995993.
[17]Li Y, Lui KO, Zhou B. Reassessing endothelial-to-mesenchymal transition in cardiovascular diseases. Nat Rev Cardiol. 2018;15(8):445-56.
[18]Miscianinov V, Martello A, Rose L, Parish E, Cathcart B, Mitić T, et al. MicroRNA-148b Targets the TGF-β Pathway to Regulate Angiogenesis and Endothelial-to-Mesenchymal Transition during Skin Wound Healing. Mol Ther. 2018;26(8):1996-2007.
[19]Pieterse E, Rother N, Garsen M, Hofstra JM, Satchell SC, Hoffmann M, et al. Neutrophil Extracellular Traps Drive Endothelial-to-Mesenchymal Transition. Arterioscler Thromb Vasc Biol. 2017;37(7):1371-9.
[20]Henderson NC, Rieder F, Wynn TA. Fibrosis: from mechanisms to medicines. Nature. 2020;587(7835):555-66.
[21]Liu Y, Liu Y, Deng J, Li W, Nie X. Fibroblast Growth Factor in Diabetic Foot Ulcer: Progress and Therapeutic Prospects. Front Endocrinol (Lausanne). 2021;12:744868.
[22]Wan R, Weissman JP, Grundman K, Lang L, Grybowski DJ, Galiano RD. Diabetic wound healing: The impact of diabetes on myofibroblast activity and its potential therapeutic treatments. Wound Repair Regen. 2021;29(4):573-81.
[23]Xie L, Zhang M, Dong B, Guan M, Lu M, Huang Z, et al. Improved refractory wound healing with administration of acidic fibroblast growth factor in diabetic rats. Diabetes research and clinical practice. 2011;93(3):396-403.
[24]Wang Z, Xie L, Ding G, Song S, Chen L, Li G, et al. Single-cell RNA sequencing of peripheral blood mononuclear cells from acute Kawasaki disease patients. Nature communications. 2021;12(1):5444.
[25]Viswanathan V, Dhamodharan U, Srinivasan V, Rajaram R, Aravindhan V. Single nucleotide polymorphisms in cytokine/chemokine genes are associated with severe infection, ulcer grade and amputation in diabetic foot ulcer. International journal of biological macromolecules. 2018;118(Pt B):1995-2000.
[26]Bermudez DM, Xu J, Herdrich BJ, Radu A, Mitchell ME, Liechty KW. Inhibition of stromal cell-derived factor-1α further impairs diabetic wound healing. J Vasc Surg. 2011;53(3):774-84.
[27]Rai V, Moellmer R, Agrawal DK. The role of CXCL8 in chronic nonhealing diabetic foot ulcers and phenotypic changes in fibroblasts: a molecular perspective. Mol Biol Rep. 2022;49(2):1565-72.
[28]Theocharidis G, Baltzis D, Roustit M, Tellechea A, Dangwal S, Khetani RS, et al. Integrated Skin Transcriptomics and Serum Multiplex Assays Reveal Novel Mechanisms of Wound Healing in Diabetic Foot Ulcers. Diabetes. 2020;69(10):2157-69.
[29]Sathvik M, Vuppuluri K, Dulipala P. The Association of the Neutrophil-Lymphocyte Ratio With the Outcome of Diabetic Foot Ulcer. Cureus. 2023;15(1):e33891.
[30]Vatankhah N, Jahangiri Y, Landry GJ, McLafferty RB, Alkayed NJ, Moneta GL, et al. Predictive value of neutrophil-to-lymphocyte ratio in diabetic wound healing. J Vasc Surg. 2017;65(2):478-83.
[31]Lou M, Luo P, Tang R, Peng Y, Yu S, Huang W, et al. Relationship between neutrophil-lymphocyte ratio and insulin resistance in newly diagnosed type 2 diabetes mellitus patients. BMC Endocr Disord. 2015;15:9.
[32]Shiny A, Bibin YS, Shanthirani CS, Regin BS, Anjana RM, Balasubramanyam M, et al. Association of neutrophil-lymphocyte ratio with glucose intolerance: an indicator of systemic inflammation in patients with type 2 diabetes. Diabetes Technol Ther. 2014;16(8):524-30.
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