Next Generation Proteomics of Human Neutrophil Granulocytes in Monogenic Disease

Background and objective

Neutrophil granulocytes (NG) are critical mediators of innate immunity and tissue regeneration. Few monogenic diseases affecting their differentiation and/or function have been described, yet a systems-biology perspective has not yet been opened. We set out to define the proteome and transcriptome of NG from healthy individuals and exome-sequenced patients aiming to develop a multi-dimensional perspective for diagnostic purposes.

Patients and methods

We analyzed highly purified peripheral blood neutrophil granulocytes from 68 healthy individuals, 6 children with severe congenital neutropenia (SCN) associated with ELANE mutations, 5 children with chronic granulomatous disease (CGD) with CYBA (2) or CYBB (3) mutations and 3 children with leukocyte adhesion deficiency (LAD, 2x ITGB2 mutation, 1x SLC35C1 mutation). In addition we analyzed proteomes of 2 children with genetically undetermined neutrophil defects. Cells were isolated from fresh venous blood using magnetic bead-based negative selection (purity >99%). Whole cell proteome analysis was done by data-independent acquisition using a Thermo Fisher QExactive HF mass spectrometer.

Results

We quantified a total of ~4300 proteins in each sample. This is the first study to analyze highly purified neutrophil granulocytes from a large cohort of healthy individuals as well as from patients with rare diseases affecting differentiation and/or function of NG. Intraindividual reproducibility was shown for one donor sampled at 3 different time points with a correlation coefficient of ~0.89. Principal component analysis demonstrated separation of the proteome of healthy and diseased cells. Interestingly, only 6 proteins constituted for 50% of the neutrophils proteome with the antimicrobial proteins DEF3A and S100A8 being the most abundant.

Stringent differential expression analysis (p<0.01) showed specific differences between the neutrophil proteome of healthy individuals and patients with monogenic diseases. In patients with LAD, only few proteins were found to be present in decreased (7 proteins) or increased (5 proteins) abundance. Of note, proteins with lowest expression levels in LAD1 (ITGB2 mutation) were the integrins ITGB2, ITGAM and ITGAX while in LAD2 (SLC35C1 mutation) integrin expression was not affected. In contrast we noticed lack of PRCP, HDHD2 and XRCC4 protein. In patients with CGD, CYBA and CYBB were decreased in expression, independent of the underlying genotype. In contrast, the other members of the NADPH oxidase complex (NCF1/2/4, Rac1/2) were not found to be differentially expressed. This suggests that both membrane-bound proteins CYBB and CYBA stabilize each other. In total, CGD neutrophils showed limited aberrations with 23 overexpressed and 11 underexpressed proteins. In striking contrast to LAD and CGD, SCN patients with ELANE mutations had a much higher number of proteins with increased (93 proteins) or decreased (47 proteins) expression levels. It is currently unknown whether these imbalances are due to the immaturity of peripheral blood neutrophils in SCN or rather secondary to unfolded protein response and cellular stress mechanisms.

In ongoing studies, we analyze the transcriptome of healthy neutrophil granulocytes in correlation to the proteome. Preliminary data indicate that there is poor correlation (PCC 0.11) between protein and mRNA levels, indicating that granule proteins are synthesized during earlier steps of differentiation.

In two patients where exome sequencing did not reveal any underlying mutation, proteome analysis showed decreased expression of NCF1 and RAB27A, respectively. In both patients we were able to use targeted sequencing of amplified DNA to show the causative mutations in NCF1 and RAB27A. Multi-omic analysis may therefore be superior to unidimensional genomic sequencing to establish a molecular diagnosis.

Conclusion

In-depth mass spectrometry-based proteomics provides new insights into disease-specific alterations of neutrophil granulocytes and may help to refine molecular diagnostics.