IL-1β targeted nanobody promotes cardiac repair through IL10-STAT3 axon-dependent M2 macrophage polarization

Acute myocardial infarction (AMI) triggers a sterile inflammatory response that drives adverse cardiac remodeling and progressive tissue injury. While interleukin-1β (IL-1β) is a recognized mediator of post-ischemic inflammation, its clinical targeting in myocardial infarction remains limited, and the underlying cardioprotective mechanisms are incompletely understood. We therefore engineered three humanized anti-IL-1β nanobodies (KD: 29.23-43.09 nM) by grafting complementarity-determining regions (CDRs) from monoclonal antibodies onto a humanized VHH scaffold, followed by structural alignment and affinity optimization. The lead candidate, 5MVZ-VHH, was assessed in a C57BL/6J mouse model of MI. Administration of 5MVZ-VHH significantly reduced systemic inflammation, as reflected by lower levels of IL-1β, TNF-α, and CXCL-10 (p < 0.05), while increasing the anti-inflammatory cytokine IL-10 (p < 0.0001). Furthermore, 5MVZ-VHH strongly suppressed apoptosis in the infarcted myocardium, as indicated by diminished caspase-3 cleavage and fewer TUNEL-positive nuclei (p < 0.01). Echocardiography and Masson's trichrome staining demonstrated improved cardiac function and attenuated fibrotic remodeling, consistent with the inhibition of IL-1β-mediated signaling. Mechanistically, 5MVZ-VHH not only enhanced angiogenesis, evidenced by elevated CD31⁺/α-SMA⁺ vessel density (p < 0.05), but also promoted macrophage polarization toward an M2-reparative phenotype (CD68⁺/CD206⁺, p < 0.01) through activation of the IL-10/STAT3 axis. Transcriptomic profiling identified 2957 differentially expressed genes (FDR < 0.05), and functional enrichment analysis indicated that IL-1β neutralization restored oxidative phosphorylation and modulated TNF and cAMP signaling pathways. Collectively, these findings show that 5MVZ-VHH confers cardioprotection via multimodal mechanisms involving coordinated inflammation suppression, apoptosis inhibition, and metabolic reprogramming, supporting its therapeutic potential for ischemic heart disease.