Single cell RNA sequencing of murine ankle joints over time reveals distinct transcriptional and cellular changes in response to Borrelia burgdorferi infection

Borreliella burgdorferi is the causative agent of Lyme disease, which is the most common vector-borne disease in the United States. While cases of Lyme disease are geographically limited to the Northeast, mid-Atlantic, and Midwest states, there are still estimates of almost 500,000 cases annually in the United States alone. In this study, we sought to assess transcriptional changes that occur in response to B. burgdorferi infection in the ankle joints of C57BL/6 mice. To this end, we performed single cell RNA sequencing (scRNA-seq) to transcriptionally profile cells, identify population heterogeneity, and analyze how cellular environments change over time. Sequencing of immune and non-immune cells in the mouse ankle joint was performed over five distinct time points, starting with uninfected animals and progressing to eight weeks post-infection in two-week increments. Overall design: Mice were inoculated subcutaneously in the center of the abdomen with 105 spirochetes in 100 µl or with 100 µl BSK-II medium for uninfected controls. B. burgdorferi infection was confirmed by culturing of live spirochetes from the ears in BSK-II medium at time of sacrifice and visualization of spirochetes using dark-field microscopy. At two, four, six and eight weeks post-infection, mice were sacrificed and ankle joints harvested. Ankle joints were processed as previously described. Briefly, joint tissue was digested for 1 hour at 37 ºC in RPMI containing 0.2 mg/ml liberase TL (Roche) and 1 unit/ml DNase (Thermo), inverting every 15 minutes. Cells were then filtered through a 40 µm filter and washed in PBS containing 2% FBS and 1 mM CaCl2. Live cells were obtained through magnetic sorting using the EasySep Dead Cell Removal (Annexin V) Kit (StemCell Technologies) per manufacturer's instructions. Following dead cell removal, cells were washed in MACS buffer (PBS containing 0.5% BSA and 2 mM EDTA) and CD45+ and CD45- cells separated using CD45 MicroBeads and a MiniMACS Separator per manufacturer's instructions (Miltenyi Biotec).