CCL24+ dermis-resident macrophages sustain Leishmania major persistence after clinical healing within a spatially segregated, type 2 immune niche

Abstract Persistent infection following clinical cure is a hallmark of Leishmania infections. It remains poorly understood despite its importance for disease reactivation and parasite transmission. Here, we identify CCL24+ dermal tissue-resident macrophages (TRMs) as the principal host cells harboring L. major in healed ear lesions of C57BL/6 mice. Histologic examination and multiplexed imaging of healed dermal lesions revealed that the infected cells were found within small, focalized areas of inflammation distinguished by the presence of infiltrating iNOS+ inflammatory monocytes, eosinophils and T cells, with high intracellular parasite loads confined to the dermal TRMs. Spatial transcriptomic analyses of healed skin confirmed the presence of small inflammatory foci characterized by monocytes, neutrophils, CD4+ and CD8+ T cells, and strong enrichment of IFN-responsive genes. Embedded within this type 1 inflammatory environment, the strongest parasite signals were localized to spatially segregated dermal niches that were enriched for type 2 associated transcripts, including Il4, Arg1, Retnla, and TRM-derived Ccl24 and Tslp. Mice with conditional deficiencies that compromised the number and M2-like activation state of the dermal TRMs, including IL-4/13-produced by eosinophils, TSLP produced by the TRMs, and IL-5-producing ILC2s, led to more efficient clearance of residual parasites, including their complete elimination from the site. Post-healing treatments, including IL-5 neutralization, or direct ablation of dermal TRMs in Ccl24-Cre:DTR mice, reduced persistent parasite burdens, in some cases to undetectable levels. Together, these findings provide direct spatial evidence that persistent pathogens can survive within localized immune-refractory tissue niches despite otherwise effective host immunity, and identify the regulatory cells and cytokines that can be targeted to achieve sterile cure.