Mast cell activation is critical for the development of allergic diseases. Ligation of sialic acid-binding immunoglobin-like lectins (Siglecs), such as Siglec-6, -7, and -8 as well as CD33, have been shown to inhibit mast cell activation. Recent studies showed that human mast cells express Siglec-9, an inhibitory receptor also expressed by neutrophils, monocytes, macrophages, and dendritic cells.
We aimed to characterize Siglec-9 expression and function in human mast cells in vitro.
We assessed the expression of Siglec-9 and Siglec-9 ligands on human mast cell lines and human primary mast cells by real-time quantitative PCR, flow cytometry, and confocal microscopy. We used a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing approach to disrupt the SIGLEC9 gene. We evaluated Siglec-9 inhibitory activity on mast cell function by using native Siglec-9 ligands, glycophorin A (GlycA), and high-molecular-weight hyaluronic acid, a monoclonal antibody against Siglec-9, and coengagement of Siglec-9 with the high-affinity receptor for IgE (FcεRI).
Human mast cells express Siglec-9 and Siglec-9 ligands. SIGLEC9 gene disruption resulted in increased expression of activation markers at baseline and increased responsiveness to IgE-dependent and IgE-independent stimulation. Pretreatment with GlycA or high-molecular-weight hyaluronic acid followed by IgE-dependent or -independent stimulation had an inhibitory effect on mast cell degranulation. Coengagement of Siglec-9 with FcεRI in human mast cells resulted in reduced degranulation, arachidonic acid production, and chemokine release.
Siglec-9 and its ligands play an important role in limiting human mast cell activation in vitro.
Copyright © 2023 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

The formation of mammalian dendritic cells (DCs) is controlled by multiple hematopoietic transcription factors, including IRF8. Loss of IRF8 exerts a differential effect on DC subsets, including plasmacytoid DCs (pDCs) and the classical DC lineages cDC1 and cDC2. In humans, cDC2-related subsets have been described including AXL+SIGLEC6+ pre-DC, DC2 and DC3. The origin of this heterogeneity is unknown. Using high-dimensional analysis, in vitro differentiation, and an allelic series of human IRF8 deficiency, we demonstrated that cDC2 (CD1c+DC) heterogeneity originates from two distinct pathways of development. The lymphoid-primed IRF8hi pathway, marked by CD123 and BTLA, carried pDC, cDC1, and DC2 trajectories, while the common myeloid IRF8lo pathway, expressing SIRPA, formed DC3s and monocytes. We traced distinct trajectories through the granulocyte-macrophage progenitor (GMP) compartment showing that AXL+SIGLEC6+ pre-DCs mapped exclusively to the DC2 pathway. In keeping with their lower requirement for IRF8, DC3s expand to replace DC2s in human partial IRF8 deficiency.
Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.

Chondrosarcoma is the second most common primary malignant bone tumor and is resistant to chemotherapy and radiation. Inadequate treatment response and poor prognosis requires novel therapeutic approaches. Proline‑rich polypeptide‑1 (PRP‑1), synthesized by brain neurosecretory cells, has demonstrated antitumor properties in JJ012‑cells; however, its underlying molecular mechanism remains unclear. The present study aimed to investigate the epigenetic regulation by which PRP‑1 inhibits chondrosarcoma cancer stem cell (CSC) proliferation and to elucidate additional CSC biomarkers in human chondrosarcoma other than ALDH1A1. Human chondrosarcoma JJ012‑cells were treated with PRP‑1 prior to performing an Aldefluor™ assay and fluorescence‑activated cell sorting in order to determine aldehyde dehydrogenase (ALDH) expression levels and isolate ALDHhigh and ALDHlow cell populations. ALDH is an established marker of CSCs in several neoplasms, including chondrosarcoma. The cells were collected and lysed for gel electrophoresis, followed by western blot analysis. The Aldefluor™ assay was used to assess the expression levels of well‑established CSC biomarkers, including CD133, CD4, CD10, CD144, CD177, CD221, CD271, leucine‑rich repeat‑containing G protein‑coupled receptor 5, SOX2 and B lymphoma Mo‑MLV insertion region 1 homolog (BMI‑1), within the ALDHhigh population of JJ012 cells. The results confirmed that ALDHA1 was the biomarker for chondrosarcoma CSCs. PRP‑1 was demonstrated to inhibit the ALDHhigh population colony and sarcosphere formation; 5 µg/ml PRP‑1 was indicated to be the optimum concentration in eliminating colonies formed by JJ012 cells (92%, P<0.001) and by the ALDHhigh CSC‑population (80.5%, P<0.001) in the clonogenic dose‑response assay. Spheroid growth unequivocally decreased with an increase in PRP‑1 dose. In order to determine the molecular mechanism by which PRP‑1 decreased the CSC population, the regulation of the mammalian Switch/sucrose non‑fermenting (SWI/SNF) complex, also referred to as BRG1‑associated factor (BAF) complex, which either activates or represses transcription, thus acting as an oncogene or tumor suppressor in human cells, was analyzed. PRP‑1 was demonstrated to decrease the expression levels of BRG, BAF170 and BRM; therefore, in JJ012 cells, these key players of the SWI/SNF (BAF) complex served an oncogenic role. The results of the present study demonstrated that PRP‑1 targets chromatin‑remodeling complexes; therefore, future efforts will be directed towards determining the interconnection between CSC maintenance, self‑renewal capacity and BAF complexes.