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Mazur et al. Rare Dis Orphan Drugs J 2023;2:1 https://dx.doi.org/10.20517/rdodj.2022.12 Page 7 of 10

complex that regulates its transport into granules, can result in neutropenia.

Although in most cases, ELANE-based neutropenia is attributed to apoptotic death of promyelocytes,
especially in the context of the UPR, mutant ELANE-dependent impaired differentiation of neutrophil
precursors has also been proposed as a mechanism of neutrophil shortage. When human and murine
myeloid cell lines with an inducible expression of human ELANE were used, suppressed expression of
[22]
transcription factors that contribute to granulocytic differentiation was observed . Since the suppression of
these factors coincided with an altered subcellular localization of NE within the cells, these findings suggest
that the mislocalization of the pathogenic NE variant may perturb the transcriptional regulation of
granulopoiesis without affecting cell viability .
[22]

NE INHIBITORS IN THE DIAGNOSIS AND TREATMENT OF NEUTROPENIA
Despite the fact that neutropenia-associated NE mutations may or may not severely reduce NE proteolytic
activity, blocking NE enzymatic activity using small synthetic NE inhibitors can correct NE localization to
primary granules, which suggests that NE inhibitors are required for the proper localization of NE and
[32]
points to the possible therapeutic or diagnostic potential of NE inhibitors . Some specific low molecular
weight inhibitors of NE have been found to positively affect the differentiation of ELANE-mutated cells.
Inhibitors such as MK0339 or Sivelestat could improve the ability of the promyelocytic cells to
[32]
differentiate . Neutropenia patient-derived iPSCs are normally able to differentiate into granulocytes only
in the presence of high doses of G-CSF, but when additionally supplemented with Sivelestat, granulocyte
differentiation was possible with a much lower dose of G-CSF; this might be potentially beneficial for future
therapies - lowering the chance of the development of myeloid malignancies. In addition, the presence of a
small NE inhibitor restored normal intracellular NE localization [18,30] . NE inhibitor MK0339 was also able to
correct the defective granulocytic differentiation of iPSCs and HL60 cells expressing mutant NE. Moreover,
the inhibitor promoted the differentiation of both iPSC cells derived either from healthy donors or SCN
[32]
patients .
Since NE and other serine proteases stored in neutrophil primary granules can contribute to tissue damage
and severe pathology such as SCN, they have to be tightly controlled. However, despite the fact that
neutrophils are a rich source of different protease inhibitors (PI), the role of these proteins in neutrophil
development and/or function remains obscure. For example, neutrophils have long been shown to produce
various PI, such as α-1 PI (SerpinA1), SLPI or elafin (PI3) [33,34] . Although proteolytically-active NE mutants,
[23]
like unmutated NE, are vulnerable to inhibition with SerpinA1 , the ability of other endogenous NE
inhibitors to control the activity of NE mutants remains largely unknown.

More recently, many more PI genes have been found to be expressed in neutrophils, including SERPINB1,
SERPINB2, SERPINB8, SERPINB9, SERPINB10, SERPINE1, SPINK1, WFDC5, and WFDC12 [35,36] . The
expression of some of these genes increases in long-lived neutrophils exposed to inflammatory stimuli, with
elafin, SERPINB9 and SLPI most highly upregulated .
[35]

Even though the expression levels of the majority of these PI in neutrophil precursors remain to be
determined, the deficiency of one of them, SLPI, has been found to be associated with SCN. SLPI expression
is strongly reduced in myeloid cells from SCN patients carrying the ELANE or HAX1 mutations . The
[37]
treatment of myeloid cells with purified non-mutated NE highly increased the expression of SLPI. Likewise,
silencing ELANE using specific shRNA has shown the dependence of SLPI expression on ELANE or HAX1
levels. These results suggest that NE is needed to induce SLPI expression in myeloid progenitors. The
knockdown of SLPI in bone marrow progenitors inhibited the G-CSF-driven formation of neutrophils by