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Borek et al. Rare Dis Orphan Drugs J 2023;2:5 https://dx.doi.org/10.20517/rdodj.2022.20 Page 3 of 14

complexity of the innate and adaptive immune cell crosstalk in disease development. Already in the late
[27]
'60s, the association of mast cells (MCs) with remodeled vessels has been shown . Notably, the depletion of
MCs in experimental PAH, as well as inhibition of their activity, attenuates pulmonary vascular
remodeling [28-31] . Regulatory T cells (Tregs), natural killer (NK) cells, and NKT cells appear to play a
beneficial role in the maintenance of vascular homeostasis and their deficiency and altered function have
been linked to PA remodeling and fibrosis [32-35] . Experimental and clinical PAH is also accompanied by
increased numbers and activation of dendritic cells (DCs) and macrophages in remodeled vessels [10,36,37] . The
development of inflammatory macrophages depends on the influx of monocytes from the circulation and
analysis of lung tissue from hypoxic mice and patients with PAH shows increased levels of factors that
promote monocyte migration (e.g., CCL1, CCL2, CX3CL1) [38,39] . Moreover, the lungs of IPAH patients show
evidence of lymphoid neogenesis. Adjacent to remodeled vessels, organized tertiary lymphoid tissues (TLT),
comprised of B and T cell zones, can be found, raising an intriguing possibility of local adaptive immune
[10]
response in these patients . Growing attention is given to the role of neutrophils in PAH pathogenesis.
Increased neutrophil to lymphocyte ratio (NLR) has been identified in the blood of the patients and NLR
correlates with prognostic PAH biomarkers. Higher NLR is associated with increased levels of N-terminal
pro-brain natriuretic peptide (NT-proBNP), rise in right atrial pressure (RAP), and shorter 6-minute
walking distance (6MWD) [40,41] . Furthermore, elevated NLR is linked to increased morbidity, mortality, and
unfavorable transplantation-free survival, independent of hemodynamic parameters and C-reactive
protein . Activated neutrophils can extrude their intracellular content of chromatin fibers and granule-
[41]
associated proteases, forming net-like structures called neutrophil extracellular traps (NETs). Under
physiological circumstances, this process (NETosis) plays a fundamental role in pathogen clearance .
[42]
However, excessive NETosis has been linked to vascular damage and inflammation . Elevated levels of
[43]
NETosis markers, i.e., DNA-myeloperoxidase complexes, neutrophil elastase, and citrullinated histone H3,
[44]
can be found both in the circulation and occlusive vascular lesions in the lungs of PAH patients .
Cumulatively, a large body of evidence supports the notion that vessel remodeling in PAH is accompanied
by an accumulation of several different types of leukocytes which mediate a broad spectrum of pro-
inflammatory actions in affected vasculature and surrounding lung tissue.

IMMUNE CELL-DERIVED SERINE PROTEASES ARE IMPORTANT REGULATORS OF
INFLAMMATION AND IMMUNITY
Immune cells are a major source of proteolytic activity in the lung. Serine proteases, abundantly expressed
by different types of immune cells, are involved in the processes related to immunity and inflammation in
[45]
various ways (reviewed by Korkmaz, et al. ). They can directly degrade ECM components (e.g., collagen,
elastin, and fibronectin), interact with protease-activated receptors (PARs), and regulate the availability and
activity of cytokines, chemokines, and growth factors . Three main serine proteases subgroups, classified
[46]
by their specific interaction with a substrate, include: trypsin-like serine proteases (e.g., tryptases and
granzymes), chymotrypsin-like serine proteases (e.g., chymases), and elastases (e.g., neutrophil elastase and
proteinase 3). Given the potentially destructive nature of serine proteases, it is not surprising that their
activity is tightly regulated through various mechanisms. Genes encoding neutrophil granule-associated
serine proteases are highly expressed only during early myelopoiesis . Furthermore, many serine proteases
[47]
are produced as inactive pro-forms (zymogens) and require additional post-translational modification steps,
such as cleavage of the N- and C-terminal domains, to become fully active. The maturation of NSPs is
[48]
mediated by other enzymes, e.g., cathepsin C (CtsC), as they do not possess autocatalytic properties .
Immune cells retain and store serine proteases in intracellular granules bound to proteoglycans. This
modulates their activity, limits access to cellular targets, and prevents extracellular leakage. Transcriptional
timing determines which proteases are packaged into specific classes of granules . Additionally, serine
[47]