Positive Regulation of Lyn Kinase by CD148 Is Required for B Cell Receptor Signaling in B1 but Not B2 B Cells.

B1 and B2 B cells differ in their ability to respond to T-cell-independent (TI) antigens. Here we report that the Src-family kinase (SFK) regulator CD148 has a unique and critical role in the initiation of B1 but not B2 cell antigen receptor signaling. CD148 loss-of-function mice were found to have defective B1 B-cell-mediated antibody responses against the T-cell-independent antigens NP-ficoll and Pneumovax 23 and had impaired selection of the B1 B cell receptor (BCR) repertoire. These deficiencies were associated with a decreased ability of B1 B cells to induce BCR signaling downstream of the SFK Lyn. Notably, Lyn appeared to be selectively regulated by CD148 and loss of this SFK resulted in opposite signaling phenotypes in B1 and B2 B cells. These findings reveal that the function and regulation of Lyn during B1 cell BCR signaling is distinct from other B cell subsets.

The abnormal signaling of the B cell receptor and co-receptors of lupus B cells.

It is easily understood that studying the physiology and pathophysiology of the BCRtriggered cascade is of importance, particularly in such diseases as systemic lupus erythematosus (SLE) that are considered by many as a "B cell disease". Even though B cells are not considered as the only players in lupus pathogenesis, and other immune and non-immune cells are certainly involved, it is the success of recent B cell-targeting treatment strategies that ascribe a critical role to the lupus B cell.

T-bet-expressing B cells contribute to the autoreactive plasma cell pool in Lyn-/- mice.

Systemic Lupus Erythematosus (SLE) is characterized by pathogenic autoantibodies against nucleic acid-containing antigens. Understanding which B-cell subsets give rise to these autoantibodies may reveal therapeutic approaches for SLE that spare protective responses. Mice lacking the tyrosine kinase Lyn, which limits B and myeloid cell activation, develop lupus-like autoimmune diseases characterized by increased autoreactive plasma cells (PCs). We used a fate-mapping strategy to determine the contribution of T-bet+ B cells, a subset thought to be pathogenic in lupus, to the accumulation of PCs and autoantibodies in Lyn-/- mice. Approximately, 50% of splenic PCs in Lyn-/- mice originated from T-bet+ cells, a significant increase compared to WT mice. In vitro, splenic PCs derived from T-bet+ B cells secreted both IgM and IgG anti-dsDNA antibodies. To determine the role of these cells in autoantibody production in vivo, we prevented T-bet+ B cells from differentiating into PCs or class switching in Lyn-/- mice. This resulted in a partial reduction in splenic PCs and anti-dsDNA IgM and complete abrogation of anti-dsDNA IgG. Thus, T-bet+ B cells make an important contribution to the autoreactive PC pool in Lyn-/- mice.

A novel lncRNA associated with the prognosis of patients with colorectal cancer resists apoptosis through the LYN/BCL-2 pathway.

PURPOSE: We found a novel lncRNA named lncAC138150.2 related to the overall survival and staging of patients with colorectal cancer (CRC) by bioinformatic analysis using data from the Cancer Genome Atlas (TCGA), and the study aimed to elucidate the function of lncAC138150.2 and underlying mechanisms. METHODS: Target molecules were knocked down by transfection with antisense oligonucleotides (ASOs), siRNAs, or lentiviruses and overexpressed by transfection with plasmids. The function of lncAC138150.2 was determined using histological, cytological, and molecular biology methods. The underlying mechanism of lncAC138150.2 function was investigated using RNA-seq, bioinformatics analysis, and molecular biology methods. RESULTS: The expression of lncAC138150.2 was increased in colorectal tissues compared with paired normal tissues. The lncAC138150.2 knockdown increased apoptosis but did not change the cell proliferation, cell cycle distribution, or cell migration ability of CRC cells, while lncAC138150.2 overexpression decreased CRC apoptosis. lncAC138150.2 was mainly located in the cell nucleus, and each lncAC138150.2 transcript knockdown increased CRC apoptosis. BCL-2 pathway was significantly altered in apoptosis induced by lncAC138150.2 knockdown, which was alleviated by BAX knockdown. The expression of LYN was significantly decreased with lncAC138150.2 knockdown, LYN knockdown increased CRC apoptosis, and its overexpression completely alleviated CRC apoptosis induced by lncAC138150.2 knockdown. CONCLUSION: lncAC138150.2 significantly inhibited CRC apoptosis and affected the prognosis of patients with CRC, through the LYN/BCL-2 pathway.

Interleukin-22 receptor 1-mediated stimulation of T-type Ca2+ channels enhances sensory neuronal excitability through the tyrosine-protein kinase Lyn-dependent PKA pathway.

BACKGROUND: Interleukin 24 (IL-24) has been implicated in the nociceptive signaling. However, direct evidence and the precise molecular mechanism underlying IL-24's role in peripheral nociception remain unclear. METHODS: Using patch clamp recording, molecular biological analysis, immunofluorescence labeling, siRNA-mediated knockdown approach and behavior tests, we elucidated the effects of IL-24 on sensory neuronal excitability and peripheral pain sensitivity mediated by T-type Ca2+ channels (T-type channels). RESULTS: IL-24 enhances T-type channel currents (T-currents) in trigeminal ganglion (TG) neurons in a reversible and dose-dependent manner, primarily by activating the interleukin-22 receptor 1 (IL-22R1). Furthermore, we found that the IL-24-induced T-type channel response is mediated through tyrosine-protein kinase Lyn, but not its common downstream target JAK1. IL-24 application significantly activated protein kinase A; this effect was independent of cAMP and prevented by Lyn antagonism. Inhibition of PKA prevented the IL-24-induced T-current response, whereas inhibition of protein kinase C or MAPK kinases had no effect. Functionally, IL-24 increased TG neuronal excitability and enhanced pain sensitivity to mechanical stimuli in mice, both of which were suppressed by blocking T-type channels. In a trigeminal neuropathic pain model induced by chronic constriction injury of the infraorbital nerve, inhibiting IL-22R1 signaling alleviated mechanical allodynia, which was reversed by blocking T-type channels or knocking down Cav3.2. CONCLUSION: Our findings reveal that IL-24 enhances T-currents by stimulating IL-22R1 coupled to Lyn-dependent PKA signaling, leading to TG neuronal hyperexcitability and pain hypersensitivity. Understanding the mechanism of IL-24/IL-22R1 signaling in sensory neurons may pave the way for innovative therapeutic strategies in pain management.

Fyn, Blk, and Lyn kinase inhibitors: A mini-review on medicinal attributes, research progress, and future insights.

Fyn, Blk, and Lyn are part of a group of proteins called Src family kinases. They are crucial in controlling cell communication and their response to the growth, changes, and immune system. Blocking these proteins with inhibitors can be a way to treat diseases where these proteins are too active. The primary mode of action of these inhibitors is to inhibit the phosphorylation of Fyn, Blk, and Lyn receptors, which in turn affects how signals pass within the cells. This review shows the structural and functional aspects of Fyn, Blk, and Lyn kinases, highlighting the significance of their dysregulation in diseases such as cancer and autoimmune disorders. The discussion encompasses the design strategies, SAR analysis, and chemical characteristics of effective inhibitors, shedding light on their specificity and potency. Furthermore, it explores the progress of clinical trials of these inhibitors, emphasizing their potential therapeutic applications.

Lyn-mediated glycolysis enhancement of microglia contributes to neuropathic pain through facilitating IRF5 nuclear translocation in spinal dorsal horn.

The pro-inflammatory phenotype of microglia usually induces neuroinflammatory reactions in neuropathic pain. Glycometabolism shift to glycolysis can promote the pro-inflammatory phenotype transition of microglia. The omics data analysis suggest a critical role for Lyn dysregulation in neuropathic pain. The present study aimed at exploring the mechanism of Lyn-mediated glycolysis enhancement of microglia in neuropathic pain. Neuropathic pain model was established by chronic constriction injury (CCI), then pain thresholds and Lyn expression were measured. Lyn inhibitor Bafetinib and siRNA-lyn knockdown were administrated intrathecally to evaluate the effects of Lyn on pain thresholds, glycolysis and interferon regulatory factor 5 (IRF5) nuclear translocation of microglia in vivo and in vitro. ChIP was carried out to observe the binding of transcription factors SP1, PU.1 to glycolytic gene promoters by IRF5 knockdown. Finally, the relationship between glycolysis and pro-inflammatory phenotype transition of microglia was evaluated. CCI led to the upregulation of Lyn expression and glycolysis enhancement in microglia of spinal dorsal horn. Bafetinib or siRNA-lyn knockdown intrathecally alleviated pain hyperalgesia, suppressed glycolysis enhancement and inhibited nuclear translocation of IRF5 in CCI mice. Also, IRF5 promoted the binding of transcription factors SP1, PU.1 to glycolytic gene promoters, and then the enhanced glycolysis facilitated the proliferation and pro-inflammatory phenotype transition of microglia and contributed to neuropathic pain. Lyn-mediated glycolysis enhancement of microglia contributes to neuropathic pain through facilitating IRF5 nuclear translocation in spinal dorsal horn.

Brain-derived neurotrophic factor regulates LYN kinase-mediated myosin light chain kinase activation to modulate nonmuscle myosin II activity in hippocampal neurons.

Myosins belong to a large superfamily of actin-dependent molecular motors. Nonmuscle myosin II (NM II) is involved in the morphology and function of neurons, but little is known about how NM II activity is regulated. Brain-derived neurotrophic factor (BDNF) is a prevalent neurotrophic factor in the brain that encourages growth and differentiation of neurons and synapses. In this study, we report that BDNF upregulates the phosphorylation of myosin regulatory light chain (MLC2), to increases the activity of NM II. The role of BDNF on modulating the phosphorylation of MLC2 was validated by using Western blotting in primary cultured hippocampal neurons. This result was confirmed by injecting BDNF into the dorsal hippocampus of mice and detecting the phosphorylation level of MLC2 by Western blotting. We further perform coimmunoprecipitation assay to confirm that this process depends on the activation of the LYN kinase through binding with tyrosine kinase receptor B, the receptor of BDNF, in a kinase activity-dependent manner. LYN kinase subsequently phosphorylates MLCK, further promoting the phosphorylation of MLC2. Taken together, our results suggest a new molecular mechanism by which BDNF regulates MLC2 activity, which provides a new perspective for further understanding the functional regulation of NM II in the nervous system.

CD36/Lyn kinase interactions within macrophages promotes pulmonary fibrosis in response to oxidized phospholipid.

Recent data from human studies and animal models have established roles for type II alveolar epithelial cell (AEC2) injury/apoptosis and monocyte/macrophage accumulation and activation in progressive lung fibrosis. Although the link between these processes is not well defined, we have previously shown that CD36-mediated uptake of apoptotic AEC2s by lung macrophages is sufficient to drive fibrosis. Importantly, apoptotic AEC2s are rich in oxidized phospholipids (oxPL), and amongst its multiple functions, CD36 serves as a scavenger receptor for oxPL. Recent studies have established a role for oxPLs in alveolar scarring, and we hypothesized that uptake and accrual of oxPL by CD36 would cause a macrophage phenotypic change that promotes fibrosis. To test this hypothesis, we treated wild-type and CD36-null mice with the oxPL derivative oxidized phosphocholine (POVPC) and found that CD36-null mice were protected from oxPL-induced scarring. Compared to WT mice, fewer macrophages accumulated in the lungs of CD36-null animals, and the macrophages exhibited a decreased accumulation of intracellular oxidized lipid. Importantly, the attenuated accrual of oxPL in CD36-null macrophages was associated with diminished expression of the profibrotic mediator, TGFß. Finally, the pathway linking oxPL uptake and TGFß expression was found to require CD36-mediated activation of Lyn kinase. Together, these observations elucidate a causal pathway that connects AEC2 injury with lung macrophage activation via CD36-mediated uptake of oxPL and suggest several potential therapeutic targets.

Anti-inflammatory effect of dictamnine on allergic rhinitis via suppression of the LYN kinase-mediated molecular signaling pathway during mast cell activation.

Mast cells (MCs) are important therapeutic targets for allergic diseases. High-affinity immunoglobulin E (IgE) Fc receptors (FcεRI) trigger abnormal activation of MCs. Allergic rhinitis (AR) is an IgE-mediated antigen inhalation reaction that occurs in the nasal mucosa. MC aggravation and dysfunction were observed during the early stages of AR pathogenesis. Herb-derived dictamnine exhibits anti-inflammatory effects. Here, we investigated the pharmacological effects of herb-derived dictamnine on IgE-induced activation of MCs and an ovalbumin (OVA)-induced murine AR model. The results indicated that dictamnine attenuated OVA-induced local allergic reactions and reduced body temperature in OVA-challenged mice with active systemic anaphylaxis. Additionally, dictamnine decreased the frequency of nasal rubbing and sneezing in an OVA-induced murine AR model. Moreover, dictamnine inhibited FcεRI-activated MC activation in a dose-dependent manner without causing cytotoxicity, reduced the activation of the tyrosine kinase LYN in LAD2 cells, and downregulated the phosphorylation of PLCγ1, IP3R, PKC, Erk1/2, and Akt, which are downstream of LYN. In conclusion, dictamnine suppressed the OVA-stimulated murine model of AR and activated IgE-induced MCs via the LYN kinase-mediated molecular signaling pathway, suggesting that dictamnine may be a promising treatment for AR.

Upregulation of the protein kinase Lyn is associated with renal injury in type 2 diabetes patients.

BACKGROUND: The role of inflammation in the pathogenesis of type 2 diabetes mellitus (T2DM) is well established. Lyn, a member of the nonreceptor protein tyrosine kinase Src family, has been reported to modulate inflammatory signaling pathways. METHODS: Lyn expression was assessed in kidney biopsies of 11 patients with diabetic kidney disease (DKD) and in kidney tissues of streptozotocin (STZ)-induced DKD mice. 102 recruited T2DM patients were divided into three groups: normoalbuminuria, microalbuminuria and macroalbuminuria. Twenty-one healthy volunteers were recruited as a control group. Clinical data, blood and urine samples of all individuals were collected for analysis. RESULTS: Lyn expression was augmented in the kidneys of DKD patients and STZ-induced diabetic mice. Compared with control and normoalbuminuria groups, both mRNA and protein expression of Lyn in peripheral blood mononuclear cells (PBMCs) in the macroalbuminuria group were significantly increased (p < .05). Elevated Lyn levels were independently related to urine albumin/urine creatinine ratio and were positively associated with key inflammatory factors, namely interleukin-1ß, monocyte chemoattractant protein-1, and tumor necrosis factor-α. Additionally, Lyn exhibited a noteworthy connection with renal tubular injury indicators, specifically urinary neutrophil gelatinase-associated lipocalin and urinary retinol binding protein. ROC curve analysis showed that Lyn could predict albuminuria in diabetic patients with an area under the curve of 0.844 (95% CI: 0.764-0.924). CONCLUSION: Lyn levels in PBMCs exhibited a positive correlation with the severity of albuminuria, renal tubular damage, and inflammatory responses. Hence, Lyn may be a compelling candidate for predicting albuminuria levels in diabetes.

B-Cell Receptor Signaling and Beyond: The Role of Igα (CD79a)/Igß (CD79b) in Normal and Malignant B Cells.

B-cell receptor (BCR) is a B cell hallmark surface complex regulating multiple cellular processes in normal as well as malignant B cells. Igα (CD79a)/Igß (CD79b) are essential components of BCR that are indispensable for its functionality, signal initiation, and signal transduction. CD79a/CD79b-mediated BCR signaling is required for the survival of normal as well as malignant B cells via a wide signaling network. Recent studies identified the great complexity of this signaling network and revealed the emerging role of CD79a/CD79b in signal integration. In this review, we have focused on functional features of CD79a/CD79b, summarized signaling consequences of CD79a/CD79b post-translational modifications, and highlighted specifics of CD79a/CD79b interactions within BCR and related signaling cascades. We have reviewed the complex role of CD79a/CD79b in multiple aspects of normal B cell biology and how is the normal BCR signaling affected by lymphoid neoplasms associated CD79A/CD79B mutations. We have also summarized important unresolved questions and highlighted issues that remain to be explored for better understanding of CD79a/CD79b-mediated signal transduction and the eventual identification of additional therapeutically targetable BCR signaling vulnerabilities.

Gender differences in Alzheimer's may be associated with TLR4-LYN expression in damage associated microglia and neuronal phagocytosis.

The role of Aß plaques and neurofibrillary tangles in Alzheimer's disease (AD) pathogenesis have recently come into question due to failure of many pharmaceutical agents targeting these deposits and detection of these misfolded proteins in normal human brains. Therefore, we investigated correlations between microglial activation and toll like receptor 4 (TLR4) and Lck/Yes novel tyrosine (LYN) kinase signaling in an AD mouse model. In this study, we used 5-6-month-old 5XFAD and wild type (WT) male and female mice. Immunohistochemistry (IHC) and flow cytometry (FC) were performed on their brains. Cognitive performance was assessed with the Barnes-Maze. IHC showed more Ab aggregation in microglia of female 5XFAD mice compared to their male counterparts. Increased co-localization of microglial TLR4 and LYN was also observed in AD more than WT and females more than males. IHC also suggests microglial phagocytosis of neurons in AD mice, which is supported by FC data. Our FC data also support the involvement of disease associated microglia (DAMs) in this process based on cytokine secretion. Cognitive assessment by the Barnes maze showed 5XFAD females performed worse than males. In this study, we investigated the relationship between microglial TLR4 and LYN kinase in 5XFAD male and females. Our data reveals a correlation between microglial TLR4 and LYN co-localization and AD pathogenesis, more in females than males. Targeting microglial TLR4 and Lyn in DAMs may offer new therapeutic opportunities in the treatment of AD.

An overview on microglial origin, distribution, and phenotype in Alzheimer's disease.

Alzheimer's disease (AD) is the most common neurodegenerative disease that is responsible for about one-third of dementia cases worldwide. It is believed that AD is initiated with the deposition of Ab plaques in the brain. Genetic studies have shown that a high number of AD risk genes are expressed by microglia, the resident macrophages of brain. Common mode of action by microglia cells is neuroinflammation and phagocytosis. Moreover, it has been discovered that inflammatory marker levels are increased in AD patients. Recent studies advocate that neuroinflammation plays a major role in AD progression. Microglia have different activation profiles depending on the region of brain and stimuli. In different activation, profile microglia can generate either pro-inflammatory or anti-inflammatory responses. Microglia defend brain cells from pathogens and respond to injuries; also, microglia can lead to neuronal death along the way. In this review, we will bring the different roles played by microglia and microglia-related genes in the progression of AD.

Investigating immunoregulatory effects of myeloid cell autophagy in acute and chronic inflammation.

Studies have highlighted a critical role for autophagy in the regulation of multiple cytokines. Autophagy inhibits the release of interleukin (IL)-1 family cytokines, including IL-1α, IL-1ß and IL-18, by myeloid cells. This, in turn, impacts the release of other cytokines by myeloid cells, as well as other cells of the immune system, including IL-22, IL-23, IL-17 and interferon-γ. Here, we assessed the impact of genetic depletion of the autophagy gene Atg7 in myeloid cells on acute and chronic inflammation. In a model of acute lipopolysaccharide-induced endotoxemia, loss of autophagy in myeloid cells resulted in increased release of proinflammatory cytokines, both locally and systemically. By contrast, loss of Atg7 in myeloid cells in the Lyn-/- model of lupus-like autoimmunity resulted in reduced systemic release of IL-6 and IL-10, with no effects on other cytokines observed. In addition, Lyn-/- mice with autophagy-deficient myeloid cells showed reduced expression of autoantibodies relevant to systemic lupus erythematosus, including anti-histone and anti-Smith protein. In vitro, loss of autophagy, through pharmacological inhibition or small interfering RNA against Becn1, inhibited IL-10 release by human and mouse myeloid cells. This effect was evident at the level of Il10 messenger RNA expression. Our data highlight potentially important differences in the role of myeloid cell autophagy in acute and chronic inflammation and demonstrate a direct role for autophagy in the production and release of IL-10 by macrophages.

Fyn and Lyn gene polymorphisms impact the risk of thyroid cancer.

Thyroid cancer is the most common malignancy of the endocrine glands, and during last couple of decades, its incidence has risen alarmingly, across the globe. Etiology of thyroid cancer is still debatable. There are a few worth mentioning risk factors which contribute to initiation of abnormalities in thyroid gland leading to cancer. Genetic instability is major risk factors in thyroid carcinogenesis. Among the genetic factors, the Src family of genes (Src, Yes1, Fyn and Lyn) have been implicated in many cancers but there is little data regarding the association of these (Src, Yes1, Fyn and Lyn) genes with thyroid carcinogenesis. Fyn and Lyn genes of Src family found engaged in proliferation, migration, invasion, angiogenesis, and metastasis in different cancers. This study was planned to examine the effect of Fyn and Lyn SNPs on thyroid cancer risk in Pakistani population in 500 patients and 500 controls. Three polymorphisms of Fyn gene (rs6916861, rs2182644 and rs12910) and three polymorphisms of Lyn gene (rs2668011, rs45587541 and rs45489500) were analyzed using Tetra-primer ARMS-PCR followed by DNA sequencing. SNP rs6916861 of Fyn gene mutant genotype (CC) showed statistically significant threefold increased risk of thyroid cancer (P < 0.0001). In case of rs2182644 of Fyn gene, mutant genotype (AA) indicated statistically significant 17-fold increased risk of thyroid cancer (P < 0.0001). Statistically significant threefold increased risk of thyroid cancer was observed in genotype AC (P < 0.0001) of Fyn gene polymorphism rs12910. In SNP rs2668011 of Lyn gene, TT genotype showed statistically significant threefold increased risk of thyroid cancer (P < 0.0001). In case of rs45587541 of Lyn gene, GA genotypes showed statistically significant 11-fold increased risk in thyroid cancer (P < 0.0001). Haplotype analysis revealed that AAATAG*, AGACAG*, AGCCAA*, AGCCAG*, CAATAG*, CGCCAG* and CGCCGA* haplotypes of Fyn and Lyn polymorphisms are associated with increased thyroid cancer risk. These results showed that genotypes and allele distribution of Fyn and Lyn are significantly linked with increased thyroid cancer risk and could be genetic adjuster for said disease.

HIV-1 Nef Induces Hck/Lyn-Dependent Expansion of Myeloid-Derived Suppressor Cells Associated with Elevated Interleukin-17/G-CSF Levels.

Human immunodeficiency virus (HIV) or simian immunodeficiency virus (SIV) infection causes myelodysplasia, anemia, and accumulation of inflammatory monocytes (CD14+ CD16+) through largely unknown cellular and molecular pathways. The mouse cells thought to be equivalent to human CD14+ CD16+ cells are CD11b+ Gr1+ myeloid-derived suppressor cells (MDSC). We used HIV transgenic (Tg) mouse models to study MDSC, namely, CD4C/Nef Tg mice expressing nef in dendritic cells (DC), pDC, CD4+ T, and other mature and immature myeloid cells and CD11c/Nef Tg mice with a more restricted expression, mainly in DC and pDC. Both Tg strains showed expansion of granulocytic and CD11b+ Gr1low/int cells with MDSC characteristics. Fetal liver cell transplantation revealed that this expansion was stroma-independent and abrogated in mixed Tg/non-Tg 50% chimera. Tg bone marrow (BM) erythroid progenitors were decreased and myeloid precursors increased, suggesting an aberrant differentiation likely driving CD11b+ Gr1+ cell expansion, apparently cell autonomously in CD4C/Nef Tg mice and likely through a bystander effect in CD11c/Nef Tg mice. Hck was activated in Tg spleen, and Nef-mediated CD11b+ Gr1+ cell expansion was abrogated in Hck/Lyn-deficient Nef Tg mice, indicating a requirement of Hck/Lyn for this Nef function. IL-17 and granulocyte colony-stimulating factor (G-CSF) were elevated in Nef Tg mice. Increased G-CSF levels were normalized in Tg mice treated with anti-IL-17 antibodies. Therefore, Nef expression in myeloid precursors causes severe BM failure, apparently cell autonomously. More cell-restricted expression of Nef in DC and pDC appears sufficient to induce BM differentiation impairment, granulopoiesis, and expansion of MDSC at the expense of erythroid maturation, with IL-17→G-CSF as one likely bystander contributor. IMPORTANCE HIV-1 and SIV infection often lead to myelodysplasia, anemia, and accumulation of inflammatory monocytes (CD14+ CD16+), with the latter likely involved in neuroAIDS. We found that some transgenic (Tg) mouse models of AIDS also develop accumulation of mature and immature cells of the granulocytic lineage, decreased erythroid precursors, and expansion of MDSC (equivalent to human CD14+ CD16+ cells). We identified Nef as being responsible for these phenotypes, and its expression in mouse DC appears sufficient for their development through a bystander mechanism. Nef expression in myeloid progenitors may also favor myeloid cell expansion, likely in a cell-autonomous way. Hck/Lyn is required for the Nef-mediated accumulation of myeloid cells. Finally, we identified G-CSF under the control of IL-17 as one bystander mediator of MDSC expansion. Our findings provide a framework to determine whether the Nef>Hck/Lyn>IL-17>G-CSF pathway is involved in human AIDS and whether it represents a valid therapeutic target.

ER Stress is Involved in Mast Cells Degranulation via IRE1α/miR-125/Lyn Pathway in an Experimental Intracerebral Hemorrhage Mouse Model.

The degranulation of mast cells accounts for the development of neuroinflammation following intracerebral hemorrhage (ICH). Inhibition of IRE1α, a sensor signaling protein related to endoplasmic reticulum stress, has been shown to exert anti-inflammatory effects in several neurological diseases. The objective of this study was to investigate the effects of IRE1α inhibition on mast cells degranulation in an ICH mouse model and to explore the contribution of miR-125/Lyn pathway in IRE1α-mediated mast cells degranulation. Male mice were subjected to ICH by intraparenchymal injection of autologous blood. STF083010, an inhibitor of IRE1α, was administered intranasally at 1 h after ICH induction. AntimiR-125 was delivered by intracerebroventricular (i.c.v.) injection prior to ICH induction to elucidate the possible mechanisms. Western blot analysis, immunofluorescence staining, neurological test, hematoma volume, brain water content, toluidine blue staining and reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) were performed. Endogenous phosphorylated IRE1α (p-IRE1α), tryptase, interleukin-17A (IL-17A), tumor necrosis factor α (TNF-α) and tryptase mRNA were increased in time dependent manner while miR-125b-2-3p was decreased after ICH. Inhibition of IRE1α, with STF083010, remarkably reduced brain water content, improved neurological function, decreased hematoma volume, upregulated the expression of miR-125b-2-3p, decreased the number of mast cells, and downregulated the protein expression of Lyn kinase, XBP1s (spliced X-box binding protein-1), tryptase, IL-17A and TNF-α. The downregulation of Lyn kinase, tryptase, IL-17A, TNF-α, and decreased mast cells number were reversed by antimiR-125. The present findings demonstrate that IRE1α inhibition attenuates mast cells degranulation and neuroinflammation, at least partially, through IRE1α/miR-125/Lyn signaling pathway after ICH.

In Vitro Validation of the Therapeutic Potential of Dendrimer-Based Nanoformulations against Tumor Stem Cells.

Tumor cells with stem cell properties are considered to play major roles in promoting the development and malignant behavior of aggressive cancers. Therapeutic strategies that efficiently eradicate such tumor stem cells are of highest clinical need. Herein, we performed the validation of the polycationic phosphorus dendrimer-based approach for small interfering RNAs delivery in in vitro stem-like cells as models. As a therapeutic target, we chose Lyn, a member of the Src family kinases as an example of a prominent enzyme class widely discussed as a potent anti-cancer intervention point. Our selection is guided by our discovery that Lyn mRNA expression level in glioma, a class of brain tumors, possesses significant negative clinical predictive value, promoting its potential as a therapeutic target for future molecular-targeted treatments. We then showed that anti-Lyn siRNA, delivered into Lyn-expressing glioma cell model reduces the cell viability, a fact that was not observed in a cell model that lacks Lyn-expression. Furthermore, we have found that the dendrimer itself influences various parameters of the cells such as the expression of surface markers PD-L1, TIM-3 and CD47, targets for immune recognition and other biological processes suggested to be regulating glioblastoma cell invasion. Our findings prove the potential of dendrimer-based platforms for therapeutic applications, which might help to eradicate the population of cancer cells with augmented chemotherapy resistance. Moreover, the results further promote our functional stem cell technology as suitable component in early stage drug development.

Novel phospho-specific monoclonal antibodies reveal differential regulation of tyrosine phosphorylation within the immunoreceptor tyrosine-based activation motif of the Fc receptor γ subunit leading to fine tuning of Syk activation.

The cytoplasmic region of the γ chain of the high-affinity receptor for IgE (FcεRI) contains a consensus sequence termed the immunoreceptor tyrosine-based activation motif (ITAM). Phosphorylation of the two tyrosine residues (N-terminal Y47 and C-terminal Y58) in the ITAM sequence is crucial for the recruitment and activation of Syk, a cytoplasmic tyrosine kinase with central signaling roles in mast cells. Using a reconstitution system in which individual tyrosine-to-phenylalanine substituted γ chains were expressed in γ-chain-deficient mast cells, we previously reported differential dephosphorylation of these tyrosines. Herein, we developed monoclonal antibodies highly specific to the phosphorylated Y47 and Y58 residues, which enables monitoring their phosphorylation under more physiological conditions. Using these antibodies, preferential dephosphorylation of Y58 following FcεRI stimulation was confirmed. Furthermore, Y58 is potentially more susceptible to phosphorylation than is Y47. Consistent with this, an in vitro kinase assay using these phospho-specific antibodies demonstrated that the Src family kinase Lyn, which is primarily responsible for ITAM phosphorylation, phosphorylates Y58 more efficiently than Y47. These results indicate that Y58 is more susceptible to dephosphorylation and phosphorylation than is Y47. Because a phosphate group on Y58 is more important for Syk binding than is a phosphate group on Y47, the preferential phosphorylation and dephosphorylation of Y58 may contribute to the fine tuning of Syk activity by promoting rapid recruitment and reducing excessive activation.