Microbial-ligand independent regulation of lymphpoiesis by NOD1

Aberrant differentiation of progenitor cells in the hematopoietic system is known to severely impact host immune responsiveness. Here we demonstrate that NOD1, a cytosolic innate sensor of bacterial peptidoglycan, also functions in murine hematopoietic cells as a major regulator of both the generation and differentiation of lymphoid progenitors as well as peripheral T lymphocyte homeostasis. We further show that NOD1 mediates these functions by facilitating STAT5 signaling downstream of hematopoietic cytokines. In steady-state, loss of NOD1 resulted in a modest but significant decrease in numbers of mature T, B and NK cells. During systemic protozoan infection, this defect was markedly enhanced leading to host mortality. Lack of functional NOD1 also impaired T-cell-dependent anti-tumor immunity while preventing colitis. These findings reveal that in addition to its classical role as a bacterial ligand receptor, NOD1 plays an important function in regulating adaptive immunity through interaction with a major host cytokine signaling pathway. Total CD4+ T lymphocytes were purified from the spleen and lymph nodes of WT, NOD1-/- or DCARD NOD1 mice with CD4 Micro beads (Miltenyi Biotec, 130-117-043) and AutoMACS (Milteny Biotec). To isolate lineageneg population, total bone marrow cells were treated with a cocktail of biotin-labelled anti-CD3, CD4, CD8, NK1.1, CD11b, CD11c, CD19, B220, Ly6G and TER-119 Ab, followed by incubation with anti-biotin macro beads (Miltenyi Biotec, #130-090-485) and separation on AutoMACS. RNA was extracted from approximately 1 million MACS-purified CD4+ T cells or Lineageneg BM cells with TRIzol (ThermoFisher Scientific, #15596026). An RNA-seq library was then prepared using 200 ng of total RNA and a TruSeq SR RNA sample prep kit (Illumina, #FC-122-1001) according to the manufacturer’s protocol. Libraries were sequenced for 50 cycles (single read) with a HiSeq 2000 (Illumina). Raw sequencing data were processed with CASAVA 1.8.2 software to convert and demultiplex BCL to FastQ.