Spleen Tyrosine Kinase Research Articles

Yiqi Kaimi prescription regulates protein phosphorylation to promote intestinal motility in slow transit constipation

Ethnopharmacological relevanceThe clinical efficacy of the Yiqi Kaimi prescription has been confirmed in slow transit constipation. However, the effects and biological mechanism of Yiqi Kaimi prescription are still unclear. Aims of the studyTo identify the effects of Yiqi Kaimi prescription on intestinal motility; To reveal the potential key targets and pathways of Yiqi Kaimi prescription for the treatment of slow transit constipation. Materials and methodsThe effects of Yiqi Kaimi prescription on slow transit constipation were investigated in a mouse model. The terminal ink propulsion experiment and fecal indocyanine green imaging was used to measure the intestinal transit time. Protein phosphorylation changes in colon tissues treated with Yiqi Kaimi prescription were detected using a Phospho Explorer antibody microarray. Bioinformatic analyses were performed using the Database for Annotation Visualization and Integrated Discovery (DAVID) and the Search Tool for the Retrieval of Interacting Genes (STRING). Western blot analysis and immunohistochemistry confirmed the observed changes in phosphorylation. Results: Yiqi Kaimi prescription significantly increased the intestinal transit rate (P < 0.05 vs. model) and reduced the time to first discharge of feces containing fecal indocyanine green imaging in mice (P < 0.05 vs. model). The administration of Yiqi Kaimi prescription induced phosphorylation changes in 41 proteins, with 9 upregulated proteins and 32 downregulated proteins. Functional classification of the phosphorylated proteins with DAVID revealed that the critical biological processes included tyrosine protein kinases, positive regulation of calcium-mediated signaling and response to muscle stretch. The phosphorylation of the spleen tyrosine kinase (SYK) at Tyr348 increased 2.19-fold, which was the most significant change. The phosphorylation level of the transcription factor p65 (RELA) at Thr505 was decreased 0.57-fold. SYK was a hub protein in the protein-protein interaction network and SYK and RELA formed the core of the secondary subnetwork. The key protein phosphorylation after treatment with Yiqi Kaimi prescription were verified by Western blot analysis and immunohistochemistry. ConclusionYiqi Kaimi prescription significantly enhanced intestinal motility. This effect was attributed to alterations in the phosphorylation levels of various target proteins. The observed changes in protein phosphorylation, including SYK and RELA, may serve as crucial factors in the treatment of slow transit constipation.

PCSK9 stimulates Syk, PKCδ, and NF-κB, leading to atherosclerosis progression independently of LDL receptor

Proprotein convertase subtilisin/kexin type-9 (PCSK9) binds to and degrades low-density lipoprotein (LDL) receptor, leading to increase of LDL cholesterol in blood. Its blockers have emerged as promising therapeutics for cardiovascular diseases. Here we show that PCSK9 itself directly induces inflammation and aggravates atherosclerosis independently of the LDL receptor. PCSK9 exacerbates atherosclerosis in LDL receptor knockout mice. Adenylyl cyclase-associated protein 1 (CAP1) is the main binding partner of PCSK9 and indispensable for the inflammatory action of PCSK9, including induction of cytokines, Toll like receptor 4, and scavenger receptors, enhancing the uptake of oxidized LDL. We find spleen tyrosine kinase (Syk) and protein kinase C delta (PKCδ) to be the key mediators of inflammation after PCSK9-CAP1 binding. In human peripheral blood mononuclear cells, serum PCSK9 levels are positively correlated with Syk, PKCδ, and p65 phosphorylation. The CAP1-fragment crystallizable region (CAP1-Fc) mitigates PCSK9-mediated inflammatory signal transduction more than the PCSK9 blocking antibody evolocumab does.

A novel natural Syk inhibitor suppresses IgE-mediated mast cell activation and passive cutaneous anaphylaxis

Spleen tyrosine kinase (Syk) plays a crucial role as a target for allergy treatment due to its involvement in immunoreceptor signaling. The purpose of this study was to identify natural inhibitors of Syk and assess their effects on the IgE-mediated allergic response in mast cells and ICR mice. A list of eight compounds was selected based on pharmacophore and molecular docking, showing potential inhibitory effects through virtual screening. Among these compounds, sophoraflavanone G (SFG) was found to inhibit Syk activity in an enzymatic assay, with an IC50 value of 2.2 μM. To investigate the conformational dynamics of the SYK-SFG system, we performed molecular dynamics simulations. The stability of the binding between SFG and Syk was evaluated using root mean square deviation (RMSD) and root mean square fluctuation (RMSF). In RBL-2H3 cells, SFG demonstrated a dose-dependent suppression of IgE/BSA-induced mast cell degranulation, with no significant cytotoxicity observed at concentrations below 10.0 μM within 24 h. Furthermore, SFG reduced the production of TNF-α and IL-4 in RBL-2H3 cells. Mechanistic investigations revealed that SFG inhibited downstream signaling proteins, including phospholipase Cγ1 (PLCγ1), as well as mitogen-activated protein kinases (AKT, Erk1/2, p38, and JNK), in mast cells in a dose-dependent manner. Passive cutaneous anaphylaxis (PCA) experiments demonstrated that SFG could reduce ear swelling, mast cell degranulation, and the expression of COX-2 and IL-4. Overall, our findings identify naturally occurring SFG as a direct inhibitor of Syk that effectively suppresses mast cell degranulation both in vitro and in vivo.

The ABCB1 and ABCG2 efflux transporters limit brain disposition of the SYK inhibitors entospletinib and lanraplenib

The highly selective Spleen Tyrosine Kinase (SYK) inhibitors entospletinib and lanraplenib disrupt kinase activity and inhibit immune cell functions. They are developed for treatment of B-cell malignancies and autoimmunity diseases. The impact of P-gp/ABCB1 and BCRP/ABCG2 efflux transporters, OATP1a/1b uptake transporters and CYP3A drug-metabolizing enzymes on the oral pharmacokinetics of these drugs was assessed using mouse models. Entospletinib and lanraplenib were orally administered simultaneously at moderate dosages (10 mg/kg each) to female mice to assess the possibility of examining two structurally and mechanistically similar drugs at the same time, while reducing the number of experimental animals and sample-processing workload. The plasma pharmacokinetics of both drugs were not substantially restricted by Abcb1 or Abcg2. The brain-to-plasma ratios of entospletinib in Abcb1a/b−/−, Abcg2−/− and Abcb1a/b;Abcg2−/− mice were 1.7-, 1.8- and 2.9-fold higher, respectively, compared to those in wild-type mice. For lanraplenib these brain-to-plasma ratios were 3.0-, 1.3- and 10.4-fold higher, respectively. This transporter-mediated restriction of brain penetration for both drugs could be almost fully inhibited by coadministration of the dual ABCB1/ABCG2 inhibitor elacridar, without signs of acute toxicity. Oatp1a/b and human CYP3A4 did not seem to affect the pharmacokinetics of entospletinib and lanraplenib, but mouse Cyp3a may limit lanraplenib plasma exposure. Unexpectedly, entospletinib and lanraplenib increased each other's plasma exposure by 2.6- to 2.9-fold, indicating a significant drug-drug interaction. This interaction was, however, unlikely to be mediated through any of the studied transporters or CYP3A. The obtained insights may perhaps help to further improve the safety and efficacy of entospletinib and lanraplenib.

DNA hypomethylation of Syk induces oxidative stress and apoptosis via the PKCβ/P66shc signaling pathway in diabetic kidney disease.

Epigenetic alterations, especially DNA methylation, have been shown to play a role in the pathogenesis of diabetes mellitus (DM) and its complications, including diabetic kidney disease (DKD). Spleen tyrosine kinase (Syk) is known to be involved in immune and inflammatory disorders. We, therefore, investigated the possible involvement of Syk promoter methylation in DKD, and the mechanisms underlying this process. Kidney tissues were obtained from renal biopsies of patients with early and advanced DKD. A diabetic mouse model (ApoE-/- DM) was generated from ApoE knockout (ApoE-/-) mice using a high-fat and high-glucose diet combined with low-dose streptozocin intraperitoneal injection. We also established an invitro model using HK2 cells. A marked elevation in the expression levels of Syk, PKCβ, and P66shc in renal tubules was observed in patients with DKD. In ApoE-/- DM mice, Syk expression and the binding of Sp1 to the Syk gene promoter were both increased in the kidney. In addition, the promoter region of the Syk gene exhibited hypomethylation. Syk inhibitor (R788) intervention improved renal function and alleviated pathologic changes in ApoE-/- DM mice. Moreover, R788 intervention alleviated oxidative stress and apoptosis and downregulated the expression of PKCβ/P66shc signaling pathway proteins. In HK2 cells, oxLDL combined with high-glucose stimulation upregulated Sp1 expression in the nucleus (compared with control and oxLDL groups), and this was accompanied by an increase in the binding of Sp1 to the Syk gene promoter. SP1 silencing downregulated the expression of Syk and inhibited the production of reactive oxygen species and cell apoptosis. Finally, PKC agonist intervention reversed the oxidative stress and apoptosis induced by Syk inhibitor (R406). In DKD, hypomethylation at the Syk gene promoter was accompanied by an increase in Sp1 binding at the promoter. As a consequence of this enhanced Sp1 binding, Syk gene expression was upregulated. Syk inhibitors could attenuate DKD-associated oxidative stress and apoptosis via downregulation of PKCβ/P66shc signaling pathway proteins. Together, our results identify Syk as a promising target for intervention in DKD.

Abstract 1185: Novel association of an autoimmune-related axis and anti-CTLA-4-based toxicities in adjuvant immunotherapy-treated melanoma patients

Abstract Intro: Immune checkpoint inhibition (ICI) for metastatic unresectable melanoma has markedly improved survival, and has shown benefit as adjuvant (AT) and neoadjuvant (NAT) therapy. Although ICI treatments confer survival benefit, they frequently induce immune-related adverse events (irAEs), which are a substantial clinical problem, particularly in AT/NAT, where ICI is used in less advanced disease to prevent recurrence. As such, the biomarkers predicting irAE risk, in particular in AT/NAT, are urgently needed to improve patient outcomes. Methods: We assessed RNA from pre-treatment peripheral CD4+ and CD8+ T cells from adjuvant clinical trial patients (CheckMate-915; n=212 patients) treated with Nivolumab (n=130) or Ipilimumab/Nivolumab (Combo) (n=82). We performed RNA-seq differential expression analysis to identify baseline differences in the immune profiles of patients with no/mild toxicity (grade 0-2) versus severe toxicity (grade 3-5). Results: Gene ontology analysis of the differentially expressed genes (DEGs) among those with severe toxicity reveals significant enrichment of immune-related pathways (IL-2, IFN-gamma, T-cell receptors, Toll-like receptors). These are shared among cell types (CD4+/CD8+) and between treatments (Nivo/Combo). From predicted DEG protein-protein interactions, we identified a treatment- and cell-type-specific expression signature associated with irAE development in CD4+ cells from Combo-treated patients. This signature, involving genes in the spleen tyrosine kinase (SYK) pathway, correctly predicts severe irAE patients with 73% accuracy (chi-squared p-value=0.001). The signature also identifies patients with mild to moderate irAEs in organ systems previously found in autoimmune traits to be associated with SYK upregulation (including skin, GI, liver, and pancreas), with 96% of all predicted patients experiencing at least one of these SYK-related toxicities, and 79% experiencing two or more. Importantly, the signature is not associated with disease recurrence (p=0.79). Conclusions: We have identified baseline expression differences in key immune pathways in peripheral T cells from ICI-treated patients with severe irAEs, and defined an SYK-related gene signature correctly identifying over half of severe irAE patients in the Combo-treated cohort. This finding aligns with our previous work linking anti-CTLA-4 irAEs with a germline variant associated with high SYK expression. This gene signature may not only serve as a baseline biomarker of severe irAEs, but it may also help guide treatment decisions, reducing toxicity while retaining efficacy, which will be particularly important in the adjuvant setting. Citation Format: Kelsey R. Monson, Robert Ferguson, Joanna Handzlik, Jiahan Xiong, Sasha Dagayev, Leah Morales, Vylyny Chat, Anabelle Bunis, Chaitra Sreenivasaiah, Sonia Dolfi, Daniel Tenney, Iman Osman, Jeffrey Weber, Tomas Kirchhoff. Novel association of an autoimmune-related axis and anti-CTLA-4-based toxicities in adjuvant immunotherapy-treated melanoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1185.

Abstract 3244: EWS-FLI1-driven upregulation of UGT3A2 unlocks drug sensitivity pathway in Ewing sarcoma

Abstract Ewing sarcoma (ES), an aggressive pediatric bone tumor, presents a clinical challenge, particularly for metastatic or relapsed disease. The hallmark genetic alteration involves the fusion of the FET RNA-binding protein, EWSR1, with an ETS transcription factor, typically FLI1, resulting in the EWS-FLI1 fusion oncoprotein. EWS-FLI1 orchestrates a unique epigenetic program, leading to the aberrant expression of genes crucial for cell-cycle regulation, DNA damage response, and cell migration. Targeting the EWS-FLI1 fusion oncoprotein is a daunting task, which has prompted us to pursue other strategies in parallel. Here, we present an innovative approach harnessing the power of phenotypic drug screens to uncover amenable pathways for targeted medicines in ES. Leveraging data from the Broad Institute’s repurposing dataset encompassing 4,518 drug molecules screened across 578 adherent cancer cell lines using the high-throughput multiplexed screening platform known as PRISM, we identified compounds with selective efficacy against ES. The top-ranking compound identified for selective ES toxicity in the screen was R112, an ATP-competitive inhibitor of Spleen Tyrosine Kinase (SYK). In low-throughput evaluation, R112 induced selective ES cell death when compared to other sarcomas. However, the reported target of R112, SYK, is a dependency in ES based on the Cancer Dependency Map (DepMap) resource. Subsequent validation of compounds identified in the PRISM screen as selectively toxic in ES revealed that while majority of them induced selective ES cell death, their reported targets were diverse and not linked to known dependencies in ES. Notably, one of the emerging compounds, luteolin, a natural compound known for its antioxidative properties, stood out and was selected for further studies alongside R112. Comprehensive genome-wide CRISPR-rescue screens identified UGT3A2, encoding a UDP glycosyltransferase, along with its solute carrier, SLC35B4, as pivotal genes that when deleted rescued both R112 and luteolin-induced cytotoxicity in ES cells. UGT3A2, a direct target gene of EWS-FLI1, demonstrated hyperexpression in EWS-FLI1-positive ES cells and no expression in most cancer and normal cells. While ES cells do not depend on UGT3A2 for survival, this abnormal elevation of UGT3A2 expression emerged as a key determinant driving their hypersensitivity to a subset of additional small molecules identified from the PRISM screen. Despite divergent mechanisms and direct targets, these drugs shared a common phenoxy motif, which UGT3A2 glycosylated. This specific EWS-FLI1-induced UGT3A2 upregulation, thus, unveils a novel biomarker for drug sensitivity,paving the way for precision medicine strategies tailored for Ewing sarcoma. Beyond Ewing sarcoma, this work holds promise in reshaping therapeutic paradigms and offering new avenues for discovering safe and enhanced treatments across a spectrum of cancers. Citation Format: Olasunkanmi Olaoye, Colleen Harrington, Kenneth Ross, Bo Kyung A. Seong, Luk de Waal, Jun Qi, Jennifer Roth, Kimberly Stegmaier. EWS-FLI1-driven upregulation of UGT3A2 unlocks drug sensitivity pathway in Ewing sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3244.

Abstract 880: Identifying ANKRD22 and fostamatinib as a theragnostic target and drug candidate for metastatic pancreatic cancer through in silico analysis

Abstract Introduction: Pancreatic cancer (PC) presents significant clinical challenges, marked by a low 5-year survival rate and frequent distant metastasis. When PC metastasizes to other organs, it leads to a notable decrease in the 5-year survival rate for PC in metastasis, dropping from 11% to 3%. Despite the effectiveness demonstrated by advanced anti-cancer therapies targeting these mutated genes, the improvement in survival rates remains modest. Another critical consideration is the rising resistance to Gemcitabine, a standard drug used for treating PC. Hence, it is imperative to gain a more profound understanding of the comprehensive mechanisms underlying metastatic pancreatic cancer (mPC) and actively explore novel drugs for its treatment. Methods: First, we reanalyzed the RNA-seq of three of PC's most common metastatic sites, including liver, lung, and peritoneum. Using the Galaxy platform, we identified DEGs (differentially expressed genes) between metastatic sites. After that, we further utilized other web-based tools and databases, such as GEPIA, TIMER 2.0, Alphafold, PubChem, Drugbank, and Chembl, to identify ANKRD22 and perform molecular docking of Fostamatinib binds to this gene. Results: Our research focused on exploring the role of ANKRD22, which contains four copies of L-shaped ankyrin motifs, an emerging oncogenic molecule associated with various cancers. In the current study, we identified that ANKRD22 exhibited significant expression in the pancreatic adenocarcinoma (PAAD) cohort, correlating with deteriorating OS rates in PC patients. Moreover, it displayed distinct expression patterns in advanced stages of the disease compared to early stages, Furthermore, we uncovered a noteworthy correlation between ANKRD22 and the Kras, TP53, CDKN2A, and SMAD4 genetic anomaly, linked to diminished survival rates, as widely recognized, in the initiation and progression of PC, these four genes stand out as the most frequently mutated. Mechanistically, ANKRD22 exhibited associations with KRAS dependency signature, U12 type spliceosome assembly, multivesicular body routing, cell invasion, tumor cell line invasion, and tumor cell line migration, all indicative of PC progression and dissemination. Expanding our investigation through the DrugBank and ChEMBL databases, we delved into fostamatinib, an FDA-approved drug known as a spleen tyrosine kinase (SYK) inhibitor. Molecular docking experiments revealed the potential interaction between ANKRD22 and fostamatinib, suggesting its candidacy to treat mPC. Conclusion: Our findings identify ANKRD22 as a pivotal oncogenic factor promoting metastasis in PC and propose fostamatinib as a potential inhibitory treatment. Citation Format: Huong Thi Luu Kim Huynh, Hendrick Gao-Min Lim, Yuan-Chii Gladys Lee, Chien-Hsin Chen, Alexander T.H. Wu. Identifying ANKRD22 and fostamatinib as a theragnostic target and drug candidate for metastatic pancreatic cancer through in silico analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 880.

FLI1 induces erythroleukemia through opposing effects on UBASH3A and UBASH3B expression

BackgroundFLI1 is an oncogenic transcription factor that promotes diverse malignancies through mechanisms that are not fully understood. Herein, FLI1 is shown to regulate the expression of Ubiquitin Associated and SH3 Domain Containing A/B (UBASH3A/B) genes. UBASH3B and UBASH3A are found to act as an oncogene and tumor suppressor, respectively, and their combined effect determines erythroleukemia progression downstream of FLI1.MethodsPromoter analysis combined with luciferase assays and chromatin immunoprecipitation (ChIP) analysis were applied on the UBASH3A/B promoters. RNAseq analysis combined with bioinformatic was used to determine the effect of knocking-down UBASH3A and UBASH3B in leukemic cells. Downstream targets of UBASH3A/B were inhibited in leukemic cells either via lentivirus-shRNAs or small molecule inhibitors. Western blotting and RT-qPCR were used to determine transcription levels, MTT assays to assess proliferation rate, and flow cytometry to examine apoptotic index.ResultsKnockdown of FLI1 in erythroleukemic cells identified the UBASH3A/B genes as potential downstream targets. Herein, we show that FLI1 directly binds to the UBASH3B promoter, leading to its activation and leukemic cell proliferation. In contrast, FLI1 indirectly inhibits UBASH3A transcription via GATA2, thereby antagonizing leukemic growth. These results suggest oncogenic and tumor suppressor roles for UBASH3B and UBASH3A in erythroleukemia, respectively. Mechanistically, we show that UBASH3B indirectly inhibits AP1 (FOS and JUN) expression, and that its loss leads to inhibition of apoptosis and acceleration of proliferation. UBASH3B also positively regulates the SYK gene expression and its inhibition suppresses leukemia progression. High expression of UBASH3B in diverse tumors was associated with worse prognosis. In contrast, UBASH3A knockdown in erythroleukemic cells increased proliferation; and this was associated with a dramatic induction of the HSP70 gene, HSPA1B. Accordingly, knockdown of HSPA1B in erythroleukemia cells significantly accelerated leukemic cell proliferation. Accordingly, overexpression of UBASH3A in different cancers was predominantly associated with good prognosis. These results suggest for the first time that UBASH3A plays a tumor suppressor role in part through activation of HSPA1B.ConclusionsFLI1 promotes erythroleukemia progression in part by modulating expression of the oncogenic UBASH3B and tumor suppressor UBASH3A.

Probucol protects against brain damage caused by intra-neural pyroptosis in rats with vascular dementia through inhibition of the Syk/Ros pathway.

Neuronal injury in chronic cerebral hypoperfusion (CCH) is the main pathogenic factor of vascular dementia (VD). Clinically, there isn't a drug specifically for VD; instead, the majority of medications used to treat Alzheimer's disease (AD) are also used to treat VD. Based on the proven anti-inflammatory and antioxidant effects of Probucol, we hypothesized that it may have therapeutic effects on VD, but more research is required to determine its exact mechanism of action. In vivo experiment: We used SD rats and most commonly used bilateral carotid artery occlusion (2-VO) in VD for modeling. After successful modeling, SD rats were given Probucol 3.5 mg/kg/day for 8 weeks to evaluate the therapeutic effect. In vitro experiment: BV-2 microglia of rats were cultured and divided into Control group and Probucol group. Each group was treated with hypoxia-hypoglycemia, hypoxia-hypoglycemia hydrogen peroxide and hypoxia-hypoglycemia hydrogen peroxide Syk inhibitor respectively. The results of immunofluorescence and Western blot showed that Probucol could significantly improve the cognitive impairment induced by CCH, and the neuronal damage was also attenuated. On the one hand, the underlying mechanism of Probucol was to reduce oxidative stress and cell apoptosis of hippocampal neurons by inhibiting the expression of phosphorylated spleen tyrosine kinase (P-Syk); On the other hand, it exerted a protective effect by reducing NLRP3-dependent cell pyroptosis and inhibiting neuroinflammation induced by microglia activation. Probucol could reduce oxidative stress and cell apoptosis by inhibiting the Syk/ROS signaling pathway, thereby improving CCH-induced cognitive impairment in vitro and in vivo.

Characterization of covalent inhibitors that disrupt the interaction between the tandem SH2 domains of SYK and FCER1G phospho-ITAM.

RNA sequencing and genetic data support spleen tyrosine kinase (SYK) and high affinity immunoglobulin epsilon receptor subunit gamma (FCER1G) as putative targets to be modulated for Alzheimer's disease (AD) therapy. FCER1G is a component of Fc receptor complexes that contain an immunoreceptor tyrosine-based activation motif (ITAM). SYK interacts with the Fc receptor by binding to doubly phosphorylated ITAM (p-ITAM) via its two tandem SH2 domains (SYK-tSH2). Interaction of the FCER1G p-ITAM with SYK-tSH2 enables SYK activation via phosphorylation. Since SYK activation is reported to exacerbate AD pathology, we hypothesized that disruption of this interaction would be beneficial for AD patients. Herein, we developed biochemical and biophysical assays to enable the discovery of small molecules that perturb the interaction between the FCER1G p-ITAM and SYK-tSH2. We identified two distinct chemotypes using a high-throughput screen (HTS) and orthogonally assessed their binding. Both chemotypes covalently modify SYK-tSH2 and inhibit its interaction with FCER1G p-ITAM, however, these compounds lack selectivity and this limits their utility as chemical tools.

Transcript Profiles of Microglia/Macrophage Cells at the Borders of Chronic Active and Subpial Gray Matter Lesions in Multiple Sclerosis.

Microglia/macrophages line the border of demyelinated lesions in both cerebral white matter and the cortex in the brains of multiple sclerosis patients. Microglia/macrophages associated with chronic white matter lesions are thought to be responsible for slow lesion expansion and disability progression in progressive multiple sclerosis, whereas those lining gray matter lesions are less studied. Profiling these microglia/macrophages could help to focus therapies on genes or pathways specific to lesion expansion and disease progression. We compared the morphology and transcript profiles of microglia/macrophages associated with borders of white matter (WM line) and subpial gray matter lesions (GM line) using laser capture microscopy. We performed RNA sequencing on isolated cells followed by immunocytochemistry to determine the distribution of translational products of transcripts increased in WM line microglia. Cells in the WM line appear activated, with shorter processes and larger cell bodies, whereas those in the GM line appear more homeostatic, with smaller cell bodies and multiple thin processes. Transcript profiling revealed 176 genes in WM lines and 111 genes in GM lines as differentially expressed. Transcripts associated with immune activation and iron homeostasis were increased in WM line microglia, whereas genes belonging to the canonical Wnt signaling pathway were increased in GM line microglia. We propose that the mechanisms of demyelination and dynamics of lesion expansion are responsible for differential transcript expression in WM lines and GM lines, and posit that increased expression of the Fc epsilon receptor, spleen tyrosine kinase, and Bruton's tyrosine kinase, play a key role in regulating microglia/macrophage function at the border of chronic active white matter lesions. ANN NEUROL 2024.

Current Understanding of Immune Thrombocytopenia: A Review of Pathogenesis and Treatment Options.

The management of immune thrombocytopenia (ITP) and the prediction of patient response to therapy still represent a significant and constant challenge in hematology. ITP is a heterogeneous disease with an unpredictable evolution. Although the pathogenesis of ITP is currently better known and its etiology has been extensively studied, up to 75% of adult patients with ITP may develop chronicity, which represents a significant burden on patients' quality of life. A major risk of ITP is bleeding, but knowledge on the exact relationship between the degree of thrombocytopenia and bleeding symptoms, especially at a lower platelet count, is lacking. The actual management of ITP is based on immune suppression (corticosteroids and intravenous immunoglobulins), or the use of thrombopoietin receptor agonists (TPO-RAs), rituximab, or spleen tyrosine kinase (Syk) inhibitors. A better understanding of the underlying pathology has facilitated the development of a number of new targeted therapies (Bruton's tyrosine kinase inhibitors, neonatal Fc receptors, strategies targeting B and plasma cells, strategies targeting T cells, complement inhibitors, and newer TPO-RAs for improving megakaryopoiesis), which seem to be highly effective and well tolerated and result in a significant improvement in patients' quality of life. The disadvantage is that there is a lack of knowledge of the predictive factors of response to treatments, which would help in the development of an optimized treatment algorithm for selected patients.

Activation of leukotriene B4 receptor 1 is a prerequisite for complement receptor 3-mediated antifungal responses of neutrophils.

Invasive fungal infections are life-threatening, and neutrophils are vital cells of the innate immune system that defend against them. The role of LTA4H-LTB4-BLT1 axis in regulation of neutrophil responses to fungal infection remains poorly understood. Here, we demonstrated that the LTA4H-LTB4-BLT1 axis protects the host against Candida albicans and Aspergillus fumigatus, but not Cryptococcus neoformans infection, by regulating the antifungal activity of neutrophils. Our results show that deleting Lta4h or Blt1 substantially impairs the fungal-specific phagocytic capacity of neutrophils. Moreover, defective activation of the spleen tyrosine kinase (Syk) and extracellular signal-related kinase (ERK1/2) pathways in neutrophils accompanies this impairment. Mechanistically, BLT1 regulates CR3-mediated, β-1,3-glucan-induced neutrophil phagocytosis, while a physical interaction with CR3 with slight influence on its dynamics is observed. Our findings thus demonstrate that the LTA4H-LTB4-BLT1 axis is essential for the phagocytic function of neutrophils in host antifungal immune response against Candida albicans and Aspergillus fumigatus.

CD11B/CD18 SIALYLATION REGULATES NEUTROPHIL TRAFFICKING AND INFLAMMATORY FUNCTION IN THE INTESTINE

Abstract Pathogen-triggered neutrophil (PMN) recruitment is critical for innate immunity, but excessive PMN influx and associated mucosal tissue damage is implicated in the pathogenesis of numerous human inflammatory disorders including inflammatory bowel disease (IBD). Critically in both ulcerative colitis and Crohn’s disease, disease flares requiring medical intervention are associated with migration of large numbers of PMNs across colonic epithelium resulting in mucosal injury/ulceration. Recent studies have shown that specific PMN glycans can be targeted to reduce transepithelial migration (TEpM) the critical final step in PMN intestinal trafficking. However the role of Sialic acid (Sia), the most abundant PMN expressed terminal glycan, in regulating PMN migration and inflammatory function in the gut is not well understood. Using sophisticated models of human and murine PMN intestinal migration, we show that blocking sialidase-mediated removal of alpha 2-3 linked sialic acid from CD11b/CD18 inhibits PMN TEpM in vitro and in vivo.Measurement of cytochrome C reduction revealed that inhibition of sialidases reduced bacterial peptide (fMLF) stimulated superoxide release by human and murine PMN. Flow cytometry analyses revealed that sialidase inhibition decreased fMLF mediated PMN degranulation and fMLF mediated CD11b/CD18 conformational activation. Highlighting the functional importance of individual Sia residues, inhibition of alpha 2-3 sialidase activity with 3’ Sialyllactose (3’SL) was found to inhibit PMN intestinal trafficking in vitro and in vivo and reduce PMN degranulation responses. Immunoblotting studies showed that sialidase inhibition deactivates spleen tyrosine kinase (Syk) and p38 MAP kinase, key intracellular mediators that signal downstream of PMN CD11b/CD18. Finally, inhibition of sialidase activity reduced adhesion of PMN to ICAM-1 (a major physiological substrate of CD11b/CD18) demonstrating that endogenous PMN sialidases regulate binding interactions between migrating PMN and intestinal epithelial receptors. Taken together data show that upon activation PMN sialidases remove alpha 2-3 Sia from CD11b/CD18 which leads to integrin activation and subsequent outside in signaling mediated by Syk to drive PMN inflammatory effector functions including migration, degranulation, and superoxide release. As such targeting CD11b/CD18 sialylation represents a promising new therapeutic strategy for reducing dysregulated PMN influx and associated mucosal tissue damage in IBD and other inflammatory disorders.

Antihypertensive Medications Can Prevent Fostamatinib-Induced Blood Pressure Elevation

Fostamatinib is a tyrosine kinase inhibitor that has been shown in clinical trials to be effective against spleen tyrosine kinase in patients suffering from rheumatoid arthritis. Fostamatinib medication was linked in clinical trials to a slight increase in systemic arterial Blood Pressure (BP). This observation is consistent with other kinase inhibitors, particularly those that obstruct VEGFR2 signaling. In conscious rats, we examined the relationship between the elevation of blood pressure caused by fostamatinib and the plasma levels of the fostamatinib-active metabolite R940406. We discovered that there was a strong correlation between changes in R940406 plasma concentration and the time course of the blood pressure effect, suggesting a direct pharmacological relationship. The experiments yielded free plasma levels of R940406 up to 346 nmol/L, which is greater than the mean peak free plasma concentration of 49 nmol/L that has been recorded in clinical settings. We beyond the clinically noted mean peak free plasma concentration of 49 nmol/L, up to 346 nmol/L. We’ve shown that there are several effective ways to reduce the blood pressure elevation caused by fostamatinib dosing, including simple drug withdrawal or condoning with a variety of common antihypertensive medications like atenolol, captopril, and nifedipine. These results provide credence to prospective strategies for preserving patient safety during fostamatinib therapy. Additionally, we have shown—using nifedipine as an example drug—that this blood pressure control was not brought about by a decrease in R940406’s plasma exposure, indicating that potential pharmacological benefits of the investigational drug may be preserved while blood pressure control is managed with the use of conventional medications.

Phase I study of the Syk inhibitor sovleplenib in relapsed or refractory mature B-cell tumors.

Sovleplenib (HMPL-523) is a selective spleen tyrosine kinase (Syk) inhibitor with antitumor activity in preclinical models of B-cell malignancy. We conducted a dose-escalation and dose-expansion phase I study of sovleplenib in patients with relapsed/refractory mature Bcell tumors. Dose escalation followed a 3+3 design; patients received oral sovleplenib (200-800 mg once daily [q.d.] or 200 mg twice daily [b.i.d.], 28-day cycles). During dose expansion, patients were enrolled into four cohorts per lymphoma classification and treated at the recommended phase 2 dose (RP2D). Overall, 134 Chinese patients were enrolled (dose escalation, n=27; dose expansion, n=107). Five patients experienced dose-limiting toxicities: one each of amylase increased (200 mg q.d.), febrile neutropenia (800 mg q.d), renal failure (800 mg q.d.), hyperuricemia and blood creatine phosphokinase increased (200 mg b.i.d.) and blood bilirubin increased and pneumonia (200 mg b.i.d.). RP2D was determined as 600 mg (>65 kg) or 400 mg (≤65 kg) q.d. The primary efficacy end point of independent review committee-assessed objective response rate in indolent B-cell lymphoma was 50.8% (95% CI, 37.5-64.1) in 59 evaluable patients at RP2D (follicular lymphoma: 60.5%, marginal zone lymphoma: 28.6%, lymphoplasmacytic lymphoma/Waldenström macroglobulinemia, 0%). The most common (≥10% patients) grade ≥3 treatment-related adverse events in the doseexpansion phase were decreased neutrophil count (29.9%), pneumonia (12.1%) and decreased white blood cell count (11.2%). Pharmacokinetic exposures increased doseproportionally with ascending dose levels from 200-800 mg, without observed saturation. Sovleplenib showed antitumor activity in relapsed/refractory B-cell lymphoma with acceptable safety. Further studies are warranted.

Apolipoprotein C3 (ApoC3) facilitates NLRP3 mediated pyroptosis of macrophages through mitochondrial damage by accelerating of the interaction between SCIMP and SYK pathway in acute lung injury

Respiratory failure caused by severe acute lung injury (ALI) is the main cause of mortality in patients with COVID-19.This study aimed to investigate the effects and underlying biological mechanism of Apolipoprotein C3 (ApoC3) in ALI.To establish an in vivo model, C57BL/6 mice were exposed by lipopolysaccharide (LPS). For the in vitro model, murine bone marrow-derived macrophages (BMDMs) or RAW264.7 cells were stimulated with LPS + adenosine triphosphate (ATP).Serum levels of ApoC3 were found to be upregulated in patients with COVID-19 or pneumonia-induced ALI. Inhibition of ApoC3 reduced lung injury in an ALI model, while overexpression of ApoC3 promoted lung injury. ApoC3 induced mitochondrial damage-mediated pyroptosis in ALI through the activation of the NOD-like receptorprotein 3 (NLRP3) inflammasome. ApoC3 recombinant protein significantly increased SCIMP expression in the lung tissue of mice models with ALI. ApoC3 also facilitated the interaction between the SLP adapter and CSK-interacting membrane protein (SCIMP) protein and Spleen tyrosine kinase (SYK) protein in the ALI model. Moreover, ApoC3 accelerated calcium-dependent reactive oxygen species (ROS) production in the ALI model. The effects of ApoC3 on pyroptosis were mitigated by the use of a pyroptosis inhibitor or an ROS inhibitor in the ALI model. Furthermore, ApoC3 activated the expression of SYK, which in turn induced NLRP3 inflammasome-regulated pyroptosis in the ALI model. METTL3 was found to mediate the m6A mRNA expression of ApoC3.Overall, our study highlights the crucial role of ApoC3 in promoting macrophage pyroptosis in ALI through calcium-dependent ROS production and NLRP3 inflammasome activation via the SCIMP-SYK pathway, providing a potential therapeutic strategy for ALI and other inflammatory diseases.

Platelet-inspired nanomedicine targeting activated neutrophils to alleviate ulcerative colitis by free radicals scavenging and controlled neutrophil swarming

Ulcerative colitis (UC) is a chronic non-specific inflammatory disease that causes inflammation and ulcers in the intestinal tract. Current therapies focus on the relief of symptoms in the short-term while it is an urgent need to achieve long-term remission of inflammation in clinics. In the current study, Platinum-doped carbon nanodot (CD) nanozymes (PtCD) with cascade enzymatic activities for superoxide dismutase/catalase (SOD/CAT) were successfully synthesized. PtCD and spleen tyrosine kinase (SYK) inhibitor piceatannol were co-loaded in PLGA and coated with platelet membrane to form a platelet-inspired nanomedicine (PM@Pic/PtCD@NP). PtCD-loaded nanoparticles (PtCD@NP) were found to co-locate with mitochondria, scavenge reactive oxygen species (ROS), and reduce the expression of pro-inflammatory cytokines. In addition, the biomimetic modification with the platelet membrane enabled PM@Pic/PtCD@NP to hitchhike neutrophils to the site of inflammation and release piceatannol to inhibit neutrophil swarming. Intravenous administration of PM@Pic/PtCD@NP partially reversed and prevented intestinal inflammation and intestinal barrier dysfunction in a murine model of colitis. The RNA-sequencing analysis revealed that gene markers of mitochondrial respiratory chain complex Ⅰ were significantly associated with PM@Pic/PtCD@NP treatment. Collectively, This study suggests that PM@Pic/PtCD@NP treatment may serve as an innovative therapeutic strategy for the long-term remission of UC.

Sinomenium acutum Modulates Platelet Aggregation and Thrombus Formation by Regulating the Glycoprotein VI-Mediated Signalosome in Mice.

Sinomenium acutum (SA) has long been used as a traditional medicine in China, Japan, and Korea to treat a wide range of diseases. It has been traditionally used to ameliorate inflammation and improve blood circulation. However, its role in platelet activation has not been thoroughly investigated. Hence, we conducted this study to assess the potential inhibitory effect of SA on platelet aggregation and thrombus formation. The antiplatelet activities of SA were evaluated by assessing platelet aggregation, granular secretion, intracellular Ca2+ mobilization, and the Glycoprotein (GP) VI-mediated signalosome. The thrombosis and bleeding time assays were used to investigate the effect of SA (orally administered at 50 and 100 mg/kg for seven days) in mice. SA treatment at concentrations of 50, 100, and 200 μg/mL significantly reduced GPVI-mediated platelet aggregation, granular secretion, and intracellular Ca2+ mobilization. Further biochemical studies revealed that SA inhibited spleen tyrosine kinase, phospholipase Cγ2, phosphatidylinositol 3-kinase, and AKT phosphorylation. Interestingly, oral administration of SA efficiently ameliorated FeCl3-induced arterial thrombus formation without prolonging the tail bleeding time. These findings suggest that SA has beneficial effects in thrombosis and hemostasis. Therefore, SA holds promise as an effective therapeutic agent for the treatment of thrombotic diseases.