Identification of stage-specific regulators for neutrophil granule biogenesis

Abstract Neutrophil granules are membrane-bound compartments crucial for host defense, mediating these leukocyte's potent antimicrobial, inflammatory, and cytotoxic responses. Mislocalization of granule proteins is a leading cause of hereditary immune disorders such as cyclic (CN) and severe congenital neutropenia (SCN). In contrast, excessive granule release contributes to inflammatory pathologies, including cytokine storms seen in severe infection. Despite their importance, the molecular mechanisms that underlie granule biogenesis remain poorly understood, limiting therapeutic progress. In this work, we identified a cohort of stage-specific regulators which mediate both functional and impaired granule biogenesis. Granule formation occurs sequentially during the differentiation of myeloblasts into mature segmented neutrophils, in a process called granulopoiesis. Previous work has established an intricate transcriptional program which induces granule protein expression at specific stages of differentiation, to allow for the formation of three different kinds of granule. The three granule subtypes—primary (azurophilic), secondary (specific), and tertiary (gelatinase)—are defined by their unique protein cargo and fusion machinery, yet all form via vesicle budding from the Golgi apparatus. Specifically, the endoplasmic reticulum synthesizes, folds, and quality checks proteins destined for granules before transporting them through the Golgi apparatus to the trans-Golgi Network (TGN), where they are sorted into the three different types of immature secretory granules. Previous work has suggested that the azurophilic granules further mature in conjunction with the endolysosomal system. However, the mechanism by which granule-destined proteins are separated from bulk TGN content and constitutively secreted proteins remain poorly understood. In spite of the physiological importance of neutrophil granules, there has yet to be a sorting receptor established for any granule protein within these cells. Our work shows that three granule-associated proteins: Serglycin (Srgn), Chromogranin A (ChgA), and Chromogranin B (ChgB), act as stage-specific regulators of granule biogenesis. Using ER-HoxB8 progenitor cells, we found that Srgn is enriched during the early stages of differentiation, colocalizing with azurophilic granule markers (MPO, ELANE) in the TGN and early endosomes. However, once matured into metamyelocytes the majority of Srgn is excluded from mature granules; relocated to a novel, non-endolysosomal membrane compartment. In contrast, ChgA and ChgB are expressed at later stages, selectively localizing with specific (lactoferrin-positive) and gelatinase (MMP9-positive) granules, respectively. In contrast to Srgn, ChgA and ChgB remain in their respective granules throughout neutrophil maturation. Given their granule-forming roles in endocrine cells, ChgA and ChgB likely drive subtype-specific cargo sorting and packaging in neutrophils. A breakdown of granulopoiesis is known to occur in promyelocytic leukemias, resulting in loss of the specific and gelatinase granules. We found that though specific granule cargo is expressed appropriately within these cells, the granule-associated proteins lose their stage specific expression and mislocalize. In these cells, ChgA is mistargeted to the azurophilic granules via an endolysosomal dependent mechanism. The specific granule proteins, instead, localize to the constitutively expressed pathway. These results indicate that mistargeting of granule proteins is driven by these granule-associated proteins, not simply timing. Notably, in Myelodysplastic Syndromes, neutrophils often present with abnormal granulation. In these cells, though capable of expressing granule proteins, we observe mislocalization of proteins from their stage-specific regulators. This may point to a common mechanism underlying failed granulopoiesis in these diseased states: the loss of granule-associated proteins. These findings establish a novel model for neutrophil granulopoiesis, identifying a new class of molecular actors which orchestrate this complex process. Further, we propose a stage-specific regulator dependent mechanism for the failed granulopoiesis, revealing potential targets for regulating these granule subtypes in disease.