Background & Aims
Inflammatory signalling mediated by microglia and other immuno-responsive cells in the central nervous system (CNS) is tightly linked to nociception, particularly its intensity, duration and resolution.(1) Central to many inflammatory signalling pathways is transcription factor NF-kB. Negative regulation of NF-kB by TNFAIP3/A20 has been characterised in a conventional immune context(2) but only in recent years has it been explored in the CNS(3,4), with limited research in pain. Mutations of A20 have been identified in human populations and mice harbouring a representative reduction-of-function mutation, exhibit an inflammatory glial phenotype.(5,6) We hypothesise negative regulation of inflammatory signalling in the CNS by A20 plays a critical role in nociception. Here we aimed to develop a hypothesis generating analysis of human brain expression data, characterise A20 in the context of pain and explore how the reduction-of-function mutation impacts nociception and analgesic response.
Methods
We have utilised parallel techniques to converge on A20 function. Using hypothesis-generating mining of the Allan Human Brain Atlas (AHBA), through density-based clustering and linear correlations we have explored gene co-expression to identify brain regions where A20 may be functionally significant. At a cellular level we have utilised microglia-like BV2 to explore the kinetics of the A20 response, comparing responses to macrophage-like RAW264.7 and primary mouse splenocytes. Cells have been stimulated with LPS, TNF and morphine, in a time and concentration dependant manor to explore different proponents of nociception. Western blotting and ELISA were used to measure A20 and cytokines respectively. Finally, with the previously characterised reduction-of-function A20 mouse strain(5,6) we are using Von-Frey and Hargreaves tests to assess mechanical and thermal nociception in the presence or absence of morphine to determine the impact of this A20 variant on nociception.
Results
The AHBA identified a significant A20 and TLR4 correlation across the brain and through clustering analysis, identified the choroid plexus as well as several key pain structures in thalamic and brainstem regions where A20 may be critically important. In BV2 microglia and RAW 264.7 we observe A20 protein expression peaks at 6h and returns to baseline over time following LPS stimulation. At a cytokine level, we observe IL-6 and TNF peak and plateau subsequent to the peak of A20 protein expression. These data are compared with primary mouse splenocytes. We also define concentration kinetics in response to LPS and TNF. Our data suggests Morphine does not induce A20 expression under the conditions tested. Finally, we will present the results from our animal model exploring the impact of the reduction-of-function A20 has on nociception and morphine efficacy in mice.
Conclusions
Using the AHBA to identify brain regions where A20 might be of functional significance, we have developed a novel means for hypothesis generation and thus direction for further experimentation. With further validation, not only with this benefit future pain research, but neurobiology more broadly. For the first time the kinetics of the A20 response in immune like cells have been characterised. By characterising the basic biology of A20 we provide beneficial insight for therapeutic targeting and have created a framework for future exploration of its negative regulation within the CNS both in the context of pain and other neurological pathologies. Here we will provide seminal research on A20 in the context of pain and for the first time will have explored the physiologically relevant reduction-of-function A20 mutation in pain.
References
1Grace, P. M., Hutchinson, M. R., Maier, S. F. & Watkins, L. R. Pathological pain and the neuroimmune interface. Nat Rev Immunol 14, 217-231, doi:10.1038/nri3621 (2014).
2Ma, A. & Malynn, B. A. A20: linking a complex regulator of ubiquitylation to immunity and human disease. Nat Rev Immunol 12, 774-785, doi:10.1038/nri3313 (2012).
3Mohebiany, A. N. et al. Microglial A20 Protects the Brain from CD8 T-Cell-Mediated Immunopathology. Cell Rep 30, 1585-1597.e1586, doi:10.1016/j.celrep.2019.12.097 (2020).
4Voet, S. et al. A20 critically controls microglia activation and inhibits inflammasome-dependent neuroinflammation. Nature Communications 9, 2036, doi:10.1038/s41467-018-04376-5 (2018).
5Zammit, N. W. et al. Denisovan, modern human and mouse TNFAIP3 alleles tune A20 phosphorylation and immunity. Nature Immunology 20, 1299-1310, doi:10.1038/s41590-019-0492-0 (2019).
6Zammit, N. W. et al. TNFAIP3 Reduction-of-Function Drives Female Infertility and CNS Inflammation. Front Immunol 13, 811525, doi:10.3389/fimmu.2022.811525 (2022).
Presenting Author
Ben Barry
Poster Authors
Ben Barry
BSc(Hons)
University of Adelaide
Lead Author
Mark Hutchinson
University of Adelaide
Lead Author
Sanam Mustafa
PhD
University of Adelaide, school of Biomedicine
Lead Author
Daniel Barratt
BSc(Hons)
University of Adelaide. Davies Livestock Research Centre.
Lead Author
Shane Grey
PhD
Garvan Institute of medical research
Lead Author
Topics
- Mechanisms: Biological-Molecular and Cell Biology