Differential Expression of Neutrophil Granule Protein Genes in Bone Marrow Myeloid Cells at the Peak and Nadir of Neutrophil Counts in Cyclic Neutropenia

Cyclic neutropenia (CyN) is a hematologic disorder in which peripheral-blood neutrophil counts show cycles at approx. 21-days intervals. The majority of CyN patients (ca. 90 %) harbor inherited mutations in the ELANE gene. The mechanism of cycling hematopoiesis downstream of ELANEmutations is unclear. In the present study we aimed to identify if there is a geterogeniety of bone marrow (BM) myeloid progenitors and granulocytic cells at the peak and nadir of the cycle of neutrophil counts.

We performed FACS analysis of BM populations in CyN patient at the peak and nadir of the cycle and revealed reduced number of CD33^high promyelocytes at the peak, as compared to the nadir neutrophil counts (6% vs 47%). Morphological examination of BM smears confirmed this observation. These data suggest differences in myeloid differentiation potential of hematopoietic cells of CyN patient during cycle. To compare the myeloid differentiation of BM cells at the peak and nadir, we performed CFU assay using BM cells isolated at these two different time points. Indeed, we found diminished capacity to produce CFU-G colonies at the peak of cycle, in comparison to the nadir (50 vs 68). This difference might be explained by the presence of different sub-populations of myeloid cells during the cycle. It was shown that the neutrophil populations can be distinguished by membrane expression of CD177, which is GPI-linked neutrophil antigen, localized primarily to the membrane of specific granules and to the plasma membrane. The proportion of CD177⁺ cells increased during neutrophil maturation in BM. Interestingly, in healthy individuals the fraction of CD177⁺ cells appeared to be constant in each individual. We evaluated the differences of CD177⁺ cell populations in CyN patients at the peak and nadir of cycle by FACS. We found that numbers of CD33⁺CD177⁺ and CD16⁺CD177⁺ populations were different during the cycle. At the peak we measured 7,1% of CD33⁺CD177⁺ cells and 83% of CD16⁺CD177⁺ cells. At the nadir 3,78%  of cells were CD33⁺CD177⁺ and 69% were CD16⁺CD177⁺.

We further performed mRNA expression analysis of CD33⁺ BM cells isolated from CyN patient at the peak and nadir of cycle and compared it to healthy individuals. We found lower mRNA expression (more than 10-fold) of CRISP3, ELANE, OLFM4, CEACAM6, MMP8, DEFA4 and LCN2 in CD33⁺ cells at the peak of the cycle comparing to the nadir. These genes encode for neutrophil granule proteins, playing an important role in the developement and function of mature neutrophils. We further confirmed differential expression of these factors in CFU colonies using BM of CyN patient isolated at the peak and nadir of the cycle: CFU-G colonies grown from cells taken at the peak of the cycle expressed less mRNA levels of granula proteins than CFU-G colonies grown from cells taken at the nadir of the cycle.

In summary, we hypothesize that the differential expression of the granule proteins is involved in the regulation of the cycle in myeloid cells in CyN. At the peak and nadir of neutrophil counts different populations (based on CD177 expression) of myeloid progenitors and neutrophils are present in the CyN BM.