Broad-spectrum antibiotics disrupt homeostatic efferocytosis [scRNA-seq]

The clearance of apoptotic cells, termed efferocytosis, is essential for tissue homeostasis and prevention of autoimmunity. Although past studies have elucidated local molecular signals that regulate homeostatic efferocytosis1 in a tissue, whether signals arising distally also regulate homeostatic efferocytosis remains elusive. Here, we find that large peritoneal macrophages (LPMs) display impaired efferocytosis in broad-spectrum antibiotics (ABX)-treated, vancomycin-treated, and germ-free mice in vivo. Mechanistically, the microbiota-derived short-chain fatty acid butyrate directly boosted efferocytosis efficiency/capacity in mouse and human macrophages, and rescued ABX-induced LPM efferocytosis defects in vivo. Bulk mRNA sequencing of butyrate-treated macrophages in vitro and single cell mRNA sequencing of LPMs isolated from ABX-treated and butyrate-rescued mice revealed regulation of efferocytosis-supportive transcriptional programs. Specifically, we found that the efferocytosis receptor T-cell immunoglobulin and mucin domain containing 4 (TIM-4, Timd4) was downregulated in LPMs of ABX-treated mice but rescued by oral butyrate and TIM-4 was required for the butyrate-induced enhancement of LPM efferocytosis capacity. LPM efferocytosis was impaired beyond withdrawal of ABX and ABX-treated mice exhibited significantly worse disease in a mouse model of lupus. Our results demonstrate that homeostatic efferocytosis relies on distal metabolic signals and suggest that defective homeostatic efferocytosis may explain link between ABX use and inflammatory disease. Overall design: Single cell RNA sequencing (scRNAseq) of total peritoneal cells isolated from wildtype untreated mice, mice treated with broad-spectrum antibiotics, and mice treated with both broad-spectrum antibiotics and oral butyrate. Each mouse (n = 2 each condition) was labeled separately by staining with MHC Class I- barcoded antibodies bearing hashtags 1-7 (BioLegend, A0301-A0307). Library prep and sequencing were performed by the Single Cell Research Initiative (SCRI) at MSKCC as follows: scRNAseq was performed on a Chromium instrument (10X Genomics) following the user guide manual for 3' v3.1. Cells were captured in droplets, then subjected to reverse transcription and cell barcoding. Following this step, emulsions were broken, and cDNA purified using Dynabead MyOne SILANE, followed by PCR amplification per manual instruction. Approximately 30,000 cells were targeted for each sample. Samples were multiplexed together on one lane of a 10X Chromium following a published cell hashing protocol69. Final libraries were sequenced on the Illumina NovaSeq S4 platform (R1 – 28 cycles, i7 – 8 cycles, R2 – 90 cycles).