Stress responses, M2 macrophages, and a distinct microbial signature in fatal intestinal acute graft-versus-host disease

Steroid-refractory intestinal acute graft-versus-host disease (aGVHD) is a frequently fatal condition with little known about mechanisms driving failed steroid responses in gut mucosa. To uncover novel molecular insights in steroid-refractory aGVHD, we compared gene expression profiles of rectosigmoid biopsies from patients at diagnosis of clinical stage 3-4 lower intestinal aGVHD (N=22), to repeat biopsies when the patients became steroid refractory (N=22), and normal controls (N=10). We also performed single gene analyses of factors associated with tolerance (programmed death ligand-1 [PDL1], indoleamine 2,3 dioxygenase [IDO1], and T cell immunoreceptor with Ig and ITIM domains [TIGIT]) and found that significantly higher expression levels of these aGVHD inhibitory genes (PDL1, IDO1, TIGIT) at aGVHD onset became decreased in the steroid-refractory state. We examined genes triggered by microbial ligands to stimulate gut repair, amphiregulin (AREG) and the aryl hydrocarbon receptor (AhR), and found that both AREG and AhR gene expression levels were increased at aGVHD onset and remained elevated in steroid-refractory aGVHD. We also identified higher expression levels of metallothioneines, metal-binding enzymes induced in stress responses, and M2 macrophage genes in steroid-refractory aGVHD. We observed no differences in T-cell subsets between onset and steroid-refractory aGVHD. Patients with a rapidly fatal course showed greater DNA damage and a distinct microbial signature at aGVHD onset, whereas patients with more prolonged survival exhibited a gene expression profile consistent with activation of Smoothened. Our results extend the paradigm beyond T cell-centric therapies for steroid-refractory GI aGVHD and highlight new mechanisms for therapeutic exploration.