Abstract
Multinucleated giant cells (MGCs) are associated with chronic inflammation and infectious diseases, but their biological characteristics in vivo remain poorly understood because intact cells are difficult to isolate from tissues. In visceral leishmaniasis, heavily parasitized, hemophagocytic MGCs are observed in the spleens of Leishmania donovani-infected mice, suggesting that these cells may contribute to parasite persistence and disease pathology. In this study, we established a method for isolating intact MGCs from the spleens of chronically infected BALB/c mice and performed transcriptomic analysis of manually collected single cells. Gentle enzymatic digestion of infected spleens enabled the recovery of large, multinucleated, F4/80-positive cells, indicating that size-based manual selection efficiently enriched for MGCs. Principal component analysis of single-cell RNA-seq data from 10 isolated MGCs showed that most MGCs formed a cluster distinct from those of naïve and infected bulk-spleen samples and RAW264.7 macrophage cells. Differential expression analysis identified genes that were upregulated during infection and further enriched in MGCs, including Ccl8, Cd63, Cebpb, and Fabp5, which are associated with macrophage activation and immune regulation. These findings provide a practical approach for isolating splenic MGCs and offer preliminary insight into the transcriptional features of L. donovani-induced MGCs in vivo.