A living therapeutic platform for localized in situ modulation of macrophage pyroptosis ameliorates GVHD while preserving GVL.

Abstract

BACKGROUND: Graft-versus-host disease (GVHD) is a major cause of morbidity and mortality following allogeneic hematopoietic stem cell transplantation, with the gastrointestinal tract being a primary target organ. Dysregulated innate immune activation, particularly within intestinal macrophages, is increasingly recognized as a key contributor to GVHD pathogenesis; however, strategies for achieving localized and controllable immunomodulation remain limited. METHODS: Single-cell transcriptomic analyses were performed to characterize macrophage-associated inflammatory programs in intestinal tissues during GVHD. A programmable living therapeutic platform was engineered using Escherichia coli Nissle 1917 to secrete outer membrane vesicles (OMVs) encapsulating BigLEN, a peptide inhibitor of NLRP3 inflammasome activation. Following oral administration, OMV-mediated delivery, inflammasome-associated signaling, and therapeutic efficacy were evaluated using in vitro macrophage assays and multiple murine GVHD models, including a graft-versus-leukemia (GVL) setting. RESULTS: Single-cell transcriptomic analysis of intestinal biopsies revealed a marked enrichment of inflammasome and pyroptosis-associated transcriptional programs in macrophages during GVHD. To locally modulate this inflammatory axis, we engineered Escherichia coli Nissle 1917 to secrete outer membrane vesicles (OMVs) encapsulating BigLEN, a peptide inhibitor of NLRP3 inflammasome activation. Following oral administration, engineered OMVs enabled effective delivery of BigLEN to lamina propria macrophages, resulting in attenuation of inflammasome activation and associated pyroptosis-related inflammatory responses. In multiple murine GVHD models, this in situ immunomodulatory strategy significantly alleviated intestinal pathology, reduced systemic inflammatory cytokine production, and improved survival. Importantly, anti-leukemia activity was preserved in a graft-versus-leukemia (GVL) setting. CONCLUSION: This study demonstrates the feasibility of a programmable, OMV-based living therapeutic platform for localized control of macrophage inflammasome activity in the gastrointestinal tract. This strategy achieves potent, local control of GVHD without overt impairment of graft-versus-leukemia (GVL) activity, and presents a novel targeted paradigm for treating gastrointestinal inflammatory disorders.