The effect of choline-rich diet on GvHD

The gut microbiome generates a wide range of bioactive metabolites, which can activate inflammatory responses, and changes in the composition of gut microbiota are associated with pathologic processes. Dietary products such as eggs, livers, dairy products, and nuts, contain choline, phosphatidylcholine, and carnitine which can be converted into circulating metabolite trimethylamine N-oxide (TMAO) by intestinal microbiota. Previously, TMAO was shown to induce vascular inflammation and endothelial dysfunction by the formation and activation of NLRP3 inflammasomes. In vivo, choline analog, 3,3-dimethyl-1-butanol (DMB), was shown to reduce TMAO concentration. However, the impact of TMAO on the pathophysiology of graft-versus-host disease (GvHD) has not been established. Kunpeng Wu and co-authors published in Blood results of a study which investigated the role of TMAO in GvHD.¹

Methods

• Age-matched C57BL/6, Nlrp3^−/−, and BALB/c male mice were used as a model for transplant induced GvHD models:
• mice were fed with the control diet or choline-supplemented diet from Day 14 and thereafter, in the presence or absence of DMB in the drinking water
• animals treated with or without TMAO were assessed for TMAO serum levels, GvHD tissue damage (tibias, ileum, colon, liver, and skin), inflammasome and cytokine induction in bone marrow-derived macrophages (BMDMs), T cell activation and survival
• The fecal microbiome was classified using 16S rRNA sequencing

Results

Impact of TMAO on the severity and mortality of GvHD mice

• Mice with GvHD had over 2-fold increased serum concentration of TMAO compared to BMT mice (7.11μM vs8μM) and increased further with daily oral TMAO administration
• GvHD mice treated with TMAO had significantly lower survival rate and higher GvHD scores than those treated with vehicle control 
• On Day 14, TMAO-treated GvHD mice also had more severe tissue damage and increased histological scores in the ileum, colon, skin, and liver

Effect of high choline diet and DMB on TMAO production and GvHD tissue damage

• TMAO concentration was significantly elevated in GvHD mice on high choline vs normal diet (16.59μM vs14μM)
• Compared to a normal diet, choline-rich diet was associated with significantly shortened survival (p < 0.05) and increased tissue damage as assessed by GvHD scores (p < 0.01)
• No change was seen in bacterial diversity and richness between mice on high choline diet vs control diet
• DMB treatment:
  • significantly reduced serum TMAO concentration in mice on the high choline diet (2.79-fold decrease)
  • significantly reduced the choline induced GvHD severity (p < 0.05)
  • prevented the choline-induced histopathological damage

Impact of TMAO on Th1 and Th17 cells

• In GvHD mice, TMAO supplementation increased T cell infiltration within BM, spleen, liver, ileum, and colon
• T cells isolated from GvHD spleen of TMAO treated mice had increased expression of IFN-γ, IL-17, T-bet, STAT3, STAT4, and RORγt
  • There was no change in mRNA expression of IL-4 and STAT6
• In vitro, TMAO treatment did not induce allogeneic CD4+ and CD8+ T cell expansion or change the expression of GvHD-related transcription factors and cytokines indicating that TMAO has an indirect effect on T cells mediated by another cell type such as macrophages

Impact of TMAO on macrophage polarization

• TMAO treatment of GvHD mice
• Significantly increased F4/80+CD11b+ macrophages when compared to a normal diet
• Increased the percentage of F4/80+CD11b+CD16/32+ M1 macrophages relative to whole population of F4/80+CD11b+
  • Enhanced infiltration of ileum, colon, and liver with M1 phenotype F4/80+ macrophages
• Increased expression of M1 signature cytokines IL-6, CXCL9, CXCL10, and IL-1β or Nlrp3 gene in splenic F4/80+ macrophages
• In vitro, TMAO significantly enhanced BMDMs mRNA expression or protein production of M1-characterized IL-1β, IL-6, TNF-α, CXCL9, and CXCL10, while no effect was seen with choline stimulation

Activation of NLRP3 inflammasome via reactive oxygen species (ROS)

• TMAO-treated BMDMs had increased expression of NLRP3, a protein complex important for M1 polarization, which was not seen after choline treatment
• IL-1β production in vitro, IL-1β cleavage was induced in TMAO-treated BMDMs
• NLRP3 inhibitor, LCY-09, suppressed TMAO induced:
  • IL-1β secretion and accumulation of cleaved caspase-1
  • mRNA expression of M1 signature cytokines and chemokines including IL-1β, IL-6, TNF-α, CXCL9, and CXCL10
• Expression of M1 signatures in Nlrp3-/- BMDMs was not enhanced after TMAO treatment
• Following a 24-hour incubation with TMAO or vehicle control in BMDMs:
  • increased production of mitochondrial ROS due to the accumulation of dysfunctional mitochondria which could be reduced with Mito-Tempo, the mitochondrion produced ROS scavenger, which also decreased NLRP3 polarization, IL-1β, caspase 1 production, and M1 signature. However, TMAO induced NLRP3 was unaffected
  • NLRP3 induction through NF-κB activation could be antagonized with triptolide, an NF-κB inhibitor, also reducing NLRP production and polarization

Importance of NLRP3 in TMAO-enhanced GvHD

• In transplant-induced GvHD models, Nlrp3^-/- GvHD mice had increased survival and no additional tissue damage compared to mice expressing WT NLRP3 when treated with TMAO.
• There was no change in relative macrophage composition or M1 phenotype induction in the spleen of Nlrp3^-/- GvHD mice with or without TMAO stimulation, indicating that TMAO induced M1 macrophage polarization cannot take place in the absence of NLRP3.

Conclusion

The study highlights the impact of diet on microbiome metabolism and GvHD. The results demonstrate that TMAO, a microbial metabolic product of a choline-rich diet, leads to M1 macrophage polarization followed by Th1 and Th17 cells activation, which stimulates GvHD progression. The authors suggest that controlling choline diet combined with a simultaneous TMAO inhibition and other strategies including additional protection with short-chain fatty acids could help control GvHD. However, further studies are needed to explore this concept as a potential treatment for patients with GvHD.

You can read more about the role of the microbiome in GvHD here.