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Basic biology of acute GvHD
dent gene expression program in activated Treg.69 Ezh2 deletion in in vivo murine Treg reprogrammed them to express an effector phenotype that could potentially be unfavorable for aGvHD.70 In vitro pharmacological EZH2 inhibition also impaired iTreg differentiation, resulting in a significantly decreased frequency of iTreg.70 Human iTreg treated with the same inhibitor were unable to maintain suppressive activity.70 However, since inhibi- tion of EZH2 potently suppressed persistence and expan- sion of effector T cells, the impact of pharmacological inhibition of EZH2 on Treg is likely context-dependent.
HDAC
Akimova et al. established that human Treg express a unique combination of HDAC compared to effector T cells, and that treatment with several different HDAC inhibitors augmented the suppressive function of Treg in vitro.71 Specific HDAC have been implicated in modulat- ing Treg function, including HDAC3 (for both nTreg and iTreg),72 HDAC9 and HDAC6. Inhibition of HDAC9 had a positive effect on FOXP3 expression and nTreg genera- tion.73 Deletion of Hdac6 or Sirt1 resulted in similar increases in FOXP3 expression and augmented nTreg function.74 Interestingly, combined pharmacological inhi- bition of HDAC6 and SIRT1 had a synergistic effect on increasing nTreg function in vivo in mice. It is likely that the two enzymes share mechanisms in their effect via the deacetylation of FOXP3.74 However, because these stud- ies were not conducted in the context of GvHD, their results should be taken as an indication for GvHD study in the future.
In the gut, butyrate and other short-chain fatty acids (SCFA) produced by commensal bacteria may also assist with the induction and maintenance of Treg in the periphery.75,76 Possessing more Lachnospiraceae- and Ruminococcaceae-family bacteria, which belong to the class Clostridia, was correlated with greater H3 acetyla- tion, a greater Treg/Th17 cell ratio and greater protection against aGvHD.77 Members of the Clostridia class produce SCFA that comprise colonocytes’ primary energy source.78 The SCFA butyrate has notable HDAC inhibito- ry activity.79 Butyrate delivery via drinking water increased peripheral Treg in mice treated with broad- spectrum antibiotics.75 Notably, this increase did not occur in mice deficient in the conserved non-coding sequence (CNS) 1 enhancer, which is part of the Foxp3 locus. Treg isolated from these mice exhibited improved suppressor function in vitro compared to antibiotic-treated mice that did not receive butyrate. Treating CD4+ T cells with butyrate during non-specific activation in vitro was also able to induce Treg, so researchers examined the effect of the treatment on Foxp3 locus deacetylation. Butyrate-treated naïve CD4+FOXP3- T cells that were non-specifically activated for 3 days showed significant increases in Foxp3 promoter and CNS 1, 2 and 3 acetyla- tion at H3K27.75
Furusawa et al. noted that feeding mice with butyrylat- ed high-amylose maize starches significantly increased differentiation of colonic Treg; these Treg were able to suppress chronic intestinal inflammation brought on by adoptive transfer of CD4+CD45RBhi cells into Rag1−/− mice.76 Through chromatin immunoprecipitation analy- sis, researchers verified that butyrate treatment increased global acetylation levels, but also acetylation at histone H3 at (i) the Foxp3 promotor region and CNS3 prior to
FOXP3 induction and (ii) CNS1 over the course of Treg differentiation.76 Though not directly related to aGvHD, these are important findings pertaining to gut inflamma- tion that should provide direction for further study.
Other regulators
Endothelial cell dysfunction, specifically the loss of endothelial cell-derived thrombomodulin, has been asso- ciated with steroid-refractory aGvHD.80 Ranjan et al. showed that thrombomodulin is essential for the gener- ation of protease-activated protein C; incubation of human T cells with activated protein C prior to their transplantation into humanized mice increased Treg fre- quency and improved xenogeneic GvHD compared to non-incubated human T cells.80 This activity was specu- lated to take place via an epigenetic pathway that has yet to be investigated.
Epigenetic programs that influence tissue injury and regeneration during acute graft-versus-host disease
The effector phase is characterized by migration and infiltration of alloreactive effector cells into aGvHD target organs and cytotoxic attack (Figure 1). Areas surrounding tissue commonly affected by aGvHD produce chemokines (e.g., CXCL9 and CXCL10) that recruit effec- tor T cells.1,11 The cells recognize major histocompatibility complex and/or minor histocompatibility antigen mis- matches and attack tissue via a cytotoxic response medi- ated by cell-surface factors and cytokines. Alloreactive effector T cells attack tissue through mechanisms that include Fas-Fas ligand interactions, perforin- and granzyme-mediated killing and TNF-a induction of cell death.1
Concurrently, tissue regeneration from both cytotoxic damage and potentially pre-allo-HSCT conditioning com- mences. Intestinal stem cells (ISC) are crucial for the regeneration of the intestinal epithelium after injury. However, ISC are also a target of effector T cells during aGvHD, causing the intestinal epithelium to be trapped in a cycle of repeated damage. Interestingly, IL-22 plays a central role in protecting the intestinal epithelium and ISC.81,82 During aGvHD, IL-23-responsive intestinal lym- phoid cells produce and secrete IL-22. However, intestinal lymphoid cells are also targeted and eliminated during disease progression, leading to IL-22 deficiency and fur- ther ISC damage.81 Regeneration can be boosted through Wnt pathway stimulation using the Wnt agonist R- Spondin1.83 Treatment with R-Spondin1 before allo- HSCT expanded ISC and treatment after transplant enhanced surviving ISC proliferation, allowing for fortifi- cation of the intestinal lumen and aGvHD inhibition.83
Little is known about epigenetic regulation of the Wnt pathway-dependent and IL-22-mediated regeneration processes in the context of GvHD. Recent studies have suggested that BMI1, a polycomb repressive complex-1 (PRC1) component important for hematopoietic stem cell renewal, is expressed in the ISC and progenitor compart- ments.84 PRC1 is known to enact its function in gene silencing via recognition of H3K27me3.85 Bmi1 knockout resulted in reduced ISC proliferation and significant increases in cell cycle regulators p16INK4a and p19ARF84.84
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