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Peer-reviewedCC BY 4.0International Journal of Molecular Sciences

Ligand-Dependent Associations of the Bovine TLR2 R152Q Variant with Neutrophil Oxidative Burst, Extracellular DNA Release, and Innate Immune Gene Expression

  • Joanna Sadowska1
  • Dawid Tobolski2
  • Zuzanna Polak3
  • Milena Krupa4
  • Roman Wójcik5
  • Paweł Brym1
5 affiliations
  1. 1Department of Animal Genetics, Faculty of Animal Bioengineering, University of Warmia and Mazury in Olsztyn, Oczapowskiego 5, 10-719 Olsztyn, Poland
  2. 2Department of Large Animal Diseases and Clinic, Institute of Veterinary Medicine, Warsaw University of Life Sciences, 02-787 Warsaw, Poland
  3. 3Department of Epizootiology, Faculty of Veterinary Medicine, University of Warmia and Mazury in Olsztyn, Oczapowskiego 13, 10-718 Olsztyn, Poland
  4. 4School of Veterinary Medicine, University College Dublin, Belfield, D04 V1W8 Dublin, Ireland
  5. 5Department of Microbiology and Clinical Immunology, Faculty of Veterinary Medicine, University of Warmia and Mazury in Olsztyn, Oczapowskiego 13, 10-719 Olsztyn, Poland
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Abstract

The bovine Toll-like receptor 2 (TLR2) R152Q substitution lies in an extracellular leucine-rich repeat domain and may alter pathogen-associated molecular pattern recognition, but its association with neutrophil function remains unknown. We genotyped 267 Polish Holstein–Friesian cows and evaluated isolated polymorphonuclear neutrophils (PMNs) after stimulation with the TLR2 agonist peptidoglycan (PGN) and lipopolysaccharide (LPS)–a well-documented PMN activator. The oxidative burst was assessed by the nitroblue tetrazolium reduction assay, extracellular double-stranded DNA release as an indirect index of neutrophil extracellular trap (NET)-associated DNA release by PicoGreen fluorescence, and the abundance of eight innate immune transcripts by RT-qPCR. Genotype frequencies were 0.59 for GG (coding for arginine), 0.39 for GA (heterozygotes), and 0.02 for AA (coding for glutamine), showing a significant deviation from Hardy–Weinberg equilibrium. In silico structural modeling predicted an allosteric destabilizing effect of the R152Q substitution. Functional responses of PMNs were both ligand- and concentration-dependent. The heterozygous GA PMNs exhibited the highest oxidative activity and extracellular dsDNA release following PGN stimulation. Conversely, AA PMNs demonstrated altered effector responses, including lower dsDNA release under LPS stimulation, accompanied by elevated basal transcript levels of recognition genes (TLR2, TLR4, NCF1, PADI4) but reduced IRAK4 expression upon stimulation. These findings suggest that the TLR2 R152Q polymorphism is associated with ligand- and concentration-dependent variation in bovine PMN functional responses. Overall, the enhanced responses in GA PMNs point toward a potential heterozygote-associated functional advantage, whereas the predicted structural destabilization and altered effector responses in the rare AA genotype (n = 3) suggest possible selective constraints against mutant homozygotes, though these latter findings remain preliminary and require confirmation in larger cohorts.

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Cite

BibTeXRIS
BibTeX
@article{sadowska2026liganddependent,
  title = {{Ligand-Dependent Associations of the Bovine TLR2 R152Q Variant with Neutrophil Oxidative Burst, Extracellular DNA Release, and Innate Immune Gene Expression}},
  author = {Joanna Sadowska and Dawid Tobolski and Zuzanna Polak and Milena Krupa and Roman Wójcik and Paweł Brym},
  journal = {International Journal of Molecular Sciences},
  year = {2026},
  month = oct,
  doi = {10.3390/ijms27219578},
  url = {https://www.mdpi.com/1422-0067/27/21/9578},
  note = {CC BY 4.0},
}
RIS
TY  - JOUR
TI  - Ligand-Dependent Associations of the Bovine TLR2 R152Q Variant with Neutrophil Oxidative Burst, Extracellular DNA Release, and Innate Immune Gene Expression
AU  - Joanna Sadowska
AU  - Dawid Tobolski
AU  - Zuzanna Polak
AU  - Milena Krupa
AU  - Roman Wójcik
AU  - Paweł Brym
PY  - 2026
DA  - 2026/10/26
JO  - International Journal of Molecular Sciences
DO  - 10.3390/ijms27219578
UR  - https://www.mdpi.com/1422-0067/27/21/9578
AB  - The bovine Toll-like receptor 2 (TLR2) R152Q substitution lies in an extracellular leucine-rich repeat domain and may alter pathogen-associated molecular pattern recognition, but its association with neutrophil function remains unknown. We genotyped 267 Polish Holstein–Friesian cows and evaluated isolated polymorphonuclear neutrophils (PMNs) after stimulation with the TLR2 agonist peptidoglycan (PGN) and lipopolysaccharide (LPS)–a well-documented PMN activator. The oxidative burst was assessed by the nitroblue tetrazolium reduction assay, extracellular double-stranded DNA release as an indirect index of neutrophil extracellular trap (NET)-associated DNA release by PicoGreen fluorescence, and the abundance of eight innate immune transcripts by RT-qPCR. Genotype frequencies were 0.59 for GG (coding for arginine), 0.39 for GA (heterozygotes), and 0.02 for AA (coding for glutamine), showing a significant deviation from Hardy–Weinberg equilibrium. In silico structural modeling predicted an allosteric destabilizing effect of the R152Q substitution. Functional responses of PMNs were both ligand- and concentration-dependent. The heterozygous GA PMNs exhibited the highest oxidative activity and extracellular dsDNA release following PGN stimulation. Conversely, AA PMNs demonstrated altered effector responses, including lower dsDNA release under LPS stimulation, accompanied by elevated basal transcript levels of recognition genes (TLR2, TLR4, NCF1, PADI4) but reduced IRAK4 expression upon stimulation. These findings suggest that the TLR2 R152Q polymorphism is associated with ligand- and concentration-dependent variation in bovine PMN functional responses. Overall, the enhanced responses in GA PMNs point toward a potential heterozygote-associated functional advantage, whereas the predicted structural destabilization and altered effector responses in the rare AA genotype (n = 3) suggest possible selective constraints against mutant homozygotes, though these latter findings remain preliminary and require confirmation in larger cohorts.
SN  - 1422-0067
ER  -