Neutrophil Extracellular Traps (NETs) in iMCD

Background:

The etiology of iMCD remains poorly understood. The cell types and cellular pathways involved in driving pathogenesis during flare are still being investigated. Recently, scRNAseq of peripheral blood mononuclear cells (PBMCs) from 3 iMCD patients revealed a strong Type I Interferon (IFN-I) gene signature and auto-antibody profiling has identified auto-antibodies in patient serum. Neutrophils are important regulators of innate and adaptive immune responses and display a number of phenotypic changes in various systemic autoimmune diseases. Unfortunately, most neutrophils are not included in PBMC samples, so very limited profiling has been done of neutrophils in iMCD. If we had evidence that they may be pathogenic, we would potentially change processing protocols to obtain/study them. 

Neutrophil extracellular traps (NETs) are net-like structures composed of DNA-histone complexes that are released by activated neutrophils, usually in response to bacteria, fungi, or viruses. NETs can be induced by auto-antibodies, and NETs are capable of inducing potent IFN-I production by plasmacytoid dendritic cells and thus a strong IFN-I response in other cells (PMC3143837). Increasing evidence suggests that NETs play an important role in autoimmunity including SLE and RA. The purpose of this study is to determine if NETs play a role in the pathogenesis/etiology of iMCD and to gain an initial indication of the potential role of neutrophils in the inflammatory/immune response in iMCD. 

Research Questions:

Goal: Use multiple measures of NETs and NET-based cellular apoptosis to determine potential involvement in iMCD pathogenesis

Important Research Questions: 

  1. Are there differences between iMCD patients and healthy controls in the amount of remnants of NETs in serum as an indicator of the amount of NETs being produced/NETosis (assayed by DNA-elastase and cit-H3-DNA)? Are there differences between iMCD flare and remission samples? What would explain a difference in results between DNA-elastase and cit-H3-DNA?

  2. Are there differences between iMCD patients and healthy controls in the ability for their serum to induce NET production (assayed by DNA (RFU, 40,000-60,000 range)? Are there differences between iMCD flare and remission samples?

  3. Are there differences between iMCD patients and healthy controls in the ability of their serum to degrade NETs (assayed by DNA (RFU, 14,000-26,000 range))? Are there differences between iMCD flare and remission samples? 

  4. Do the initial NET-based findings correlate with clinical measures for these patients surrounding sample collection? (ex: Albumin levels are negatively associated with formation of NETs)

  5. Decreased Albumin in flare =  increased NETs? Or higher CHA score = higher NETs?

  6. Do the 3 patients with IFN-I signatures from scRNAseq tend to have a particular NET profile? 

  7. Are there any correlations that can be made between auto-antibody profiles from Utz and UCSF studies and the NET profiles in this study?