Heterogeneity of neutrophil states in rheumatoid arthritis and systemic lupus

• Applicant: Professor Dr. Ricardo Grieshaber-Bouyer
• Subject Area: Rheumatology
• Term: since 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 517717827

Neutrophils are phenotypically and functionally heterogenous and play an important pathophysiologic role in rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). However, it is unclear which heterogeneity neutrophils display in RA and SLE on the single cell level and how specific effector functions in neutrophils can be targeted selectively. We recently demonstrated that murine neutrophils in homeostasis are organized along the chronological neutrotime sequence and reach distinct polarization states in experimental inflammation. We developed an approach to jointly analyze murine and human transcriptomic data. Thereby, we identified a highly conserved gene expression program which characterizes neutrophils in arthritic joints in mice and humans. Based on this groundwork, we hypothesize that neutrophils in RA and SLE display shared as well as disease-specific polarization states. Further, we hypothesize that perturbation of genes dysregulated in inflammation will disclose which targets selectively impact one function and which targets impact global neutrophil function. In aim 1, we will dissect which polarization states neutrophils display in RA and SLE on the single cell level using CITE-seq. We will integrate these new data with published data and perform a cross-species analysis of neutrophils in inflammation. For all polarization states, we will identify differentially expressed genes and active regulatory networks, signaling pathways and transcription factors. In aim 2, we will evaluate the impact of genetic perturbations on neutrophil function. We will transduce cell lines of myeloid progenitors (murine HoxB8 and human HL-60 cells) with a library of gRNAs against the top differentially expressed genes and predicted transcription factors shared across human and murine inflamed neutrophils. We will differentiate these myeloid precursors and test migration, phagocytosis and ROS production in vitro. We will sort the most and least functionally competent neutrophils in each assay, sequence the sgRNA locus and identify the functional impact of individual genes. Finally, we will adoptively transfer three ER-HoxB8 knockout clones cells into irradiated mice and test the impact of individual genes on experimental peritonitis and K/BxN serum transfer arthritis. Supported by our preliminary data, both aims can be performed in parallel. Together, this revised first-time proposal will systematically dissect neutrophil heterogeneity in RA and SLE, the conservation of neutrophil polarization states across humans and mice and mechanistically examine which genes influence different functional parameters in neutrophils.

• DFG Programme: Research Grants