Defining human skin immunity to cutaneous leishmaniasis via systems immunology approaches

National Institute Of Allergy And Infectious Diseases

Investigators

Abstract

(1) Construction of a single cell transcriptomic atlas of human cutaneous leishmaniasis. We continued our collaborative project with Dr. Elise O'Connell (LPD) and the NIH Leishmaniasis Clinic to expand on our group's single cell transcriptomic atlas of human CL. Over the past year, we evaluated nine patients of whom seven were confirmed to have Leishmania infection by PCR of a skin punch biopsy from a CL lesion. This year, we added two additional cases of L. braziliensis, as well as five cases from infections with three Leishmania species not yet represented in the atlas (one patient with L. major, one patient with L. panamensis, and three patients with L. mexicana). From each of these patients, we obtained one skin punch biopsy from a CL lesion. Additionally, this year Dr. O'Connell received NIH IRB approval for a protocol amendment to begin collecting a negative control punch biopsy from each patient of unaffected healthy skin from an anatomically matched site on the side contralateral to the biopsied lesion. Lesion and healthy skin biopsies were dissociated into a single cell suspension and assayed by droplet-based single cell RNA-seq (scRNA-seq). Last year, the atlas contained 140,000 sequenced cells; this year, we added an additional 93,000 sequenced single cells, comprised of 87,000 CL lesion cells and 6,000 healthy skin cells for a total atlas size of 233,000 cells. Bioinformatic analysis is being performed in collaboration with Drs. Justin Lack and Jian Sun (NIAID/RTB/IDSS). Analysis of the scRNA-seq data using Seurat revealed 17 cell clusters which were mapped to the Human Skin Cell Atlas using the ScType algorithm. The largest cell clusters are cytotoxic T cells (21%) and T helper cells (15%). Other cell subsets identified in the atlas include Tregs, ILC1/NKs, macrophages, dendritic cells, keratinocytes, fibroblasts, vascular endothelial cells, pericytes, mast cells, and melanocytes. In comparison to matched healthy skin controls, stromal cells in CL lesions show a marked shift towards inflammatory gene expression, particularly in keratinocytes, fibroblasts, and vascular endothelial cells. The scRNA-seq data highlight potentially novel intercellular circuits between stromal and immune cells that drive immunopathology in CL lesions. Additional scRNA-seq libraries are currently being constructed comprising an estimated 560,000 CL lesion cells and 332,000 patient-matched healthy skin cells. These libraries, along with single cell TCR-seq libraries to track T cell clonotypes, will be sequenced and analyzed in the coming months. (2) Microbiopsies of human cutaneous leishmaniasis lesions. We established a collaboration with Dr. Tarl Prow (University of York, UK) to use skin microbiopsy devices to perform serial sampling of CL lesions, with the goal of tracking the dynamics of skin gene expression over the course of CL lesion treatment and healing. This year we continued to collect and bank microbiopsy samples from both the lesion and matched healthy skin (n = 5 from each site) from the seven CL patients described above. All patients tolerated the procedure well without complications and with normal healing of all the microbiopsy puncture sites. Each microbiopsy collects 50 ng of skin tissue, yielding 1 ng of either RNA or DNA for downstream studies. With protocols and technical guidance from Dr. Prow, we plan to perform a pilot study of RNA isolation and bulk RNA-seq with a subset of the microbiopsy samples. (3) Development of a multiscale tissue model of early Leishmania skin infection. We established a collaboration with Dr. James Glazier (Indiana University, Biocomplexity Institute) to develop a multiscale tissue model of the early immune response to Leishmania infection in the skin. The model was developed using CompuCell3D, a computational modeling environment that models cell movement and behavior in tissues using a Cellular Potts model. The cellular behaviors in our model were designed based on multiple in vivo imaging studies of early Leishmania infection and the interactions of parasites with immune cells in the skin. Programmed with only a sparse set of rules for intercellular interaction, chemotaxis, parasite replication, phagocytosis, and cell death, the model recapitulates the major cellular behaviors described in studies of early Leishmania skin infection. Chief among these is the Trojan horse model whereby Leishmania-infected neutrophils undergo apoptosis, resulting in infected blebs being phagocytosed by macrophages, leading to their infection. Infected macrophages then die by either necrosis or apoptosis, releasing free parasites or infected blebs that subsequently infect neighboring macrophages, leading to spread of the infection throughout the tissue. This is the first multiscale tissue model of Leishmania infection and demonstrates the utility of this computational modeling approach to generate novel experimental predictions and to identify critical early parameters that regulate the outcome of Leishmania skin infection.