The role of microglia in CNS GvHD

Graft-versus-host disease (GvHD) is a complication observed in patients following allogeneic hematopoietic cell transplantation (allo-HCT). Primarily believed to be restricted to the skin, gastrointestinal tract and liver, pre-clinical and clinical observations have since revealed the effect of GvHD on the central nervous system (CNS).¹

T cell CNS infiltration is a key player in CNS GvHD establishment and progression. As a growing concern in the field of allo-HCT, efforts have been made to determine the mechanisms behind T cell infiltration, neuronal apoptosis and subsequent neurological deficits.² Microglia are a tissue macrophage population found in the CNS and stand as the main major histocompatibility complex class II (MHC-II)-expressing antigen presenting cell (APC) in the CNS.³ Although microglia are important in mediating CNS immune responses, they have also been linked to the pathogenesis of a number of neurodegenerative diseases.⁴ A team led by Mathew R Nimitha and Vinnakota M Janaki, University of Freiburg, Freiburg, DE, investigated microglial activation in CNS GvHD mouse models and patient samples.²

Study design

• The bone marrow (BM) of experimental mice was irradiated and GvHD was induced by transplantation of BM cells and CD4⁺/CD8⁺ T cells from a mismatched donor (allo-HCT). As a control, syngeneic stem cell transplantation (syn-HCT) was performed by transplantation of BM and T cells from a donor mouse matching the genetic background of the recipient mouse
• CNS immune infiltration and activation were measured using:
  • Immunohistochemistry (IHC) and flow cytometric (FC) quantification of CD3⁺ T cells
  • IHC and FC quantification of CD11b⁺CD45^high cells (dendritic cells, monocytes, macrophages)
  • IHC quantification of Iba-1, a microglial activation marker
  • IHC and FC quantification of MHC II- and CD80-expressing microglia
  • Transcriptome analysis of Map3k7, which encodes for transforming growth factor beta (TGF-β)-activated kinase 1 (TAK 1)
  • IHC quantification of phosphorylated (p) microglia via the markers: TAK 1, pJNK, pNF-kB, pMAPK
• The elevated plus maze and novel object recognition tests were implemented to evaluate neurocognitive function in mice
• GvHD was induced in Cx3cr1^creER: Tnf^fl/- mice to investigate the role of tumour necrosis factor (TNF) in CNS tissue damage
• GvHD was induced in Cx3cr1^creER:Tak1^fl/fl to investigate the role of microglial TAK1 in CNS-GvHD
• The effect of two TAK1 inhibitors, takinib and 5Z-7-Oxozeaenol, on CNS GvHD-presenting mice was investigated
• Formalin fixed paraffin embedded (FFPE) patient brain samples (Institute of Neuropathology, University Hospital Freiburg, Germany) were obtained from patients that had died following allo-HCT and were either asymptomatic or had developed GvHD

Results

• The abundance of CD3⁺ T cells was significantly increased in the meninges (p< 0.005) and cortex (p= 0.001) of mice that had undergone allo-HCT but not after syn-HCT
• The abundance of CD11b⁺CD45^high cells was significantly greater (p= 0.005) in mice that had undergone allo-HCT compared to syn-HCT
• The abundance of Iba-1⁺ microglia cells was increased in mice that had undergone allo-HCT compared to syn-HCT (p= 0.01)
• Features of microglial activation (reduced filament dendrite length, reduced number of dendrite segments, branching points and dendrite terminal points) were observed in allo-HCT treated, GvHD-developing mice but not in syn-HCT treated or untreated mice
• Significant upregulation of MHC-II (p< 0.0001) and CD80 (p< 0.0001) on CD11b⁺CD45^high cells was observed in allo-HCT treated mice compared to syn-HCT treated or untreated mice
• In mice that developed GvHD, p38 MAPK phosphorylation was increased, indicating activation of the MAPK/NF-κB pathway
• TNF knockout (KO) mice exhibited reduced number of CD3⁺ T cells in the CNS
• NF-κB-mediated TAK1 activation was driven by TNF in mice
• Microglial TAK1 KO mice exhibited reduced of CD3⁺ cell in the CNS, reduced microglial MHC-II expression and improved neurocognitive function while maintaining peripheral GvHD presentation
• Pharmaceutical inhibition of TAK1 in mice resulted in reduced:
  • CNS CD3⁺ T cells and Iba-1⁺ microglia
  • CNS Th1 and Th17 cells
  • MHC-II expression by microglia
  • TNF production by microglia
  • Endothelial vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) expression on CNS endothelial cells
• In-vivo pharmaceutical inhibition of TAK1 improved neurocognitive function in mice
• The number of Iba-1⁺ microglia was increased in human patients who developed acute GvHD (aGvHD) following allo-HCT compared to those who did not
• The number of TNF and Iba-1 double positive microglia was significantly increased in patients who developed aGvHD compared to those who did not

Conclusions

• Microglia play a crucial role in the establishment of CNS GvHD; augmenting T-cell stimulation and migration into the CNS
• The microglial TAK1/TNF/MHC-II axis is imperative in the initiation and maintenance of CNS GvHD
• The data from this study provides the grounds for investigating TAK1 inhibition in phase I clinical trials for the treatment of CNS GvHD