Elucidating granulocytic myeloid-derived suppressor cell (G-MDSC) fate during S. aureus biofilm infection
Myeloid-derived suppressor cells (MDSCs) are pathologically activated immature myeloid cells with immunosuppressive activity that expand during chronic inflammation, such as cancer and prosthetic joint infection (PJI). MDSCs can be broadly separated into two populations based on surface marker expression and function, namely monocytic MDSCs (M-MDSCs) and granulocytic MDSCs (G-MDSCs). In cancer models, M-MDSCs have been reported to transition into tumor-associated macrophages and/or dendritic cells, whereas G-MDSCs are considered terminally differentiated with short half-lives. G-MDSCs are the most abundant leukocyte infiltrate during PJI; however, how this population is maintained in vivo and cellular heterogeneity is currently unknown. In this study, we identified a population of Ly6G+Ly6C+F4/80+MHCII+ MDSCs during PJI that displayed immunosuppressive properties ex vivo. We leveraged F4/80 and MHCII expression by these cells for further characterization using cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq), which revealed a distinct transcriptomic signature of this population. F4/80+MHCII+ MDSCs displayed gene signatures resembling G-MDSCs, neutrophils, and monocytes, but had increased expression of genes involved in cytokine response/production, inflammatory cell death, and mononuclear cell differentiation. To determine whether F4/80+MHCII+ MDSCs represented an alternative differentiation state of G-MDSCs, Ly6G+Ly6C+F4/80-MHCII- G-MDSCs from CD45.1 mice were adoptively transferred into CD45.2 recipients using a mouse model of PJI. A small percentage of transferred G-MDSCs acquired F4/80 and MHCII expression in vivo, suggesting some degree of plasticity in this population. Collectively, these results demonstrate a previously unappreciated phenotype of G-MDSCs during PJI, which suggests a novel granulocytic-to-monocytic transition of MDSC infiltrates. Overall design: A mouse model of S. aureus prosthetic joint infection was used for this study. Implant-associated tissue was collected from WT mice at day 14 following S. aureus PJI, homogenized, and after red blood cells lysis, Fc receptors were blocked. Samples were stained with CD45-APC, Ly6G-PE, Ly6C-PerCP-Cy5.5, and Live/Dead fixable blue stain, whereupon live Ly6G+Ly6C+ and Ly6C+Ly6G- cells were collected using a BD Biosciences FACSAria. Next, these populations were labeled using TotalSeq-B0114 F4/80 (RRID: AB_2819847) and TotalSeq-B0117 I-A/I-E (MHCII; RRID: AB_2832367) barcoded antibodies (BioLegend) following the manufacturer's protocol. The post-sort viability of Ly6C+Ly6G- and Ly6G+Ly6C+ populations was 79.6 and 89.4%, respectively, with minimal debris as determined using a Luna automated fluorescent cell counter (Logos Biosystems). Ly6G+Ly6C+ and Ly6C+G- cells (13,627 and 1,624, respectively) were processed using a 10X Genomics platform by the UNMC Genomics Core. Briefly, single cells were lysed, RNA was reverse-transcribed and barcoded using a Chromium Single-Cell 3' Reagent Kit v3 (10X Genomics) according to the manufacturer's protocol. Illumina compatible cDNA libraries were created and quantified by quantitative PCR using the KAPA Library Quant Kit (Illumina) and were loaded at a concentration of 1.3 pM on an Illumina NovaSeq instrument. Samples were sequenced following the parameters suggested by 10X Genomics to an average read depth of 100,000 reads/cell. Alignment was completed using 10X Genomics Cell Ranger with the Mus musculus mm10 genome as a reference. ADT; Barcode sequence TotalSeq-B0114 F4/80; TTAACTTCAGCCCGT TotalSeq-B0117 I-A/I-E; GGTCACCAGTATGAT