Introduction: Most transplants are eventually lost to chronic rejection in which macrophages are often a dominant cell type in the grafts. However, mechanisms that regulate the inflammatory features of macrophages in transplant rejection remain poorly defined. We examined the role of the epigenetic reader BRD4 in regulation of macrophage functions in transplant models and how changes in macrophage phenotypes affect transplant outcomes.
Methods: We used in vitro macrophage gene profiling and in vivo heart transplant models to study how the epigenetic reader BRD4 modulate macrophage phenotypes and transplant survival.
Results: We found that inhibition of BRD4 using the chemical inhibitor JQ1, which prevents BRD4 from binding active gene enhancers, strongly suppressed the induction of inflammatory macrophages and most prominently M2 macrophages. Further analysis by RNA-seq showed that the induction of M1 and M2-associated transcription factors was markedly suppressed upon blocking BRD4 functions, demonstrating that BRD4 controls the induction of inflammatory programs in macrophages. Indeed, ChIP-qPCR experiments showed that binding of M2-related transcription factors at the Arg-1 locus was markedly reduced after JQ1 treatment, and the expression of Arg-1 enhancer RNA (eRNA) was also abolished after BRD4 blockade. Studies using gene knockout mice showed that the nuclear localization of p-STAT6 was profoundly reduced after JQ1 treatment. Importantly, in a fully MHC mismatched heart transplant model (BALB/c to B6), we found that treatment of recipient mice with a low dose of CTLA4-Ig inhibited the acute allograft rejection, which allowed the development of chronic rejection characterized by extensive macrophage infiltration in the grafts. Strikingly, additional treatment of the transplant recipients with the BRD4 inhibitor JQ1 induced long-term heart allograft survival (>100 days) with no histological signs of chronic rejection. Analysis of graft-infiltrating cells showed that the most striking effect of JQ1 treatment was the inhibition of inflammatory macrophages in vivo.
Conclusions: Our data suggest that targeting the epigenetic reader BRD4 represents a novel strategy in abolishing chronic allograft rejection and the underlying mechanism for induction of allograft tolerance is the suppression of inflammatory macrophages.