Syk Expression Research Articles

A Novel TLR4-SYK Interaction Axis Plays an Essential Role in the Innate Immunity Response in Bovine Mammary Epithelial Cells.

Mammary gland epithelium, as the first line of defense for bovine mammary gland immunity, is crucial in the process of mammary glands' innate immunity, especially that of bovine mammary epithelial cells (bMECs). Our previous studies successfully marked SYK as an important candidate gene for mastitis traits via GWAS and preliminarily confirmed that SYK expression is down-regulated in bMECs with LPS (E. coli) stimulation, but its work mechanism is still unclear. In this study, for the first time, in vivo, TLR4 and SYK were colocalized and had a high correlation in mastitis mammary epithelium; protein-protein interaction results also confirmed that there was a direct interaction between them in mastitis tissue, suggesting that SYK participates in the immune regulation of the TLR4 cascade for bovine mastitis. In vitro, TLR4 also interacts with SYK in LPS (E. coli)-stimulated or GBS (S. agalactiae)-infected bMECs, respectively. Moreover, TLR4 mRNA expression and protein levels were little affected in bMECsSYK- with LPS stimulation or GBS infection, indicating that SYK is an important downstream element of the TLR4 cascade in bMECs. Interestingly, IL-1β, IL-8, NF-κB and NLRP3 expression in LPS-stimulated or GBS-infected bMECsSYK- were significantly higher than in the control group, while AKT1 expression was down-regulated, implying that SYK could inhibit the IL-1β, IL-8, NF-κB and NLRP3 expression and alleviate inflammation in bMECs with LPS and GBS. Taken together, our solid evidence supports that TLR4/SYK/NF-κB signal axis in bMECs regulates the innate immunity response to LPS or GBS.

Effects of n-butanol extract of Pulsatilla decoction on the NLRP3 inflammasome in macrophages infected with Candida albicans

Ethnopharmacological relevancePulsatilla decoction is a traditional Chinese medicine from Shang Han Lun that has been reported to have therapeutic efficacy in vulvovaginal candidiasis (VVC), and is a growth inhibitor of Candida albicans (C. albicans) in vitro, the causative agent of VVC.Aim of the study: In previous studies, Pulsatilla decoction has shown therapeutic benefits for VVC. With further chemical extraction of the decoction, the n-butanol extract (of Pulsatilla decoction; BEPD) was most effective against C. albicans and therapeutic for VVC. The mechanism, however, has not been elucidated. The regulation of NOD-like receptor protein 3 (NLRP3) inflammasome has recently been demonstrated as a critical component of the inflammasome complex that initiates the vaginal inflammatory response. Therefore, the effect of BEPD on NLRP3 associated with VVC was investigated. Materials and methodsUltra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used for detecting the principal compounds of BEPD (Anemoside B4, Esculin, esculetin, Epiberberine, Berberine, Jatrorrhizine and Phellodendrine). BEPD-containing serum is prepared by intragastric administration of BEPD (4.6875 g/kg for seven days) in rats. PMA-induced THP-1 cells were challenged with C. albicans. The expression of CD68 to identify macrophages was examined by flow cytometry, the viability of THP-1 cells were assessed by CCK8 assay, the release of lactate dehydrogenase (LDH) was detected by LDH kit, and the secretion levels of IL-1β and IL-18 were evaluated through enzyme-linked immunosorbent assay (ELISA), the levels of NLRP3 were quantified by immunofluorescence, the levels of reactive oxygen species (ROS) were measured by ROS kit, and the expression of Dectin-1, Syk, TLR2, TLR4, MyD88, NF-κB, NLRP3, Caspase-1, and ASC proteins were detected by Western blot. ResultsAfter administration of BEPD-containing serum, the levels of IL-1β, IL-18 and LDH released from macrophages were reduced in the BEPD-containing serum group compared to the model group. In addition, BEPD-containing serum inhibited the expression of ROS in macrophages, suppressed the expression of NLRP3 and inhibited the expression of TLRs/MyD88 and Dectin-1/Syk signaling pathway-related proteins. ConclusionsBEPD suppressed the NLRP3 inflammasome and related signaling pathways in macrophages infected with C. albicans in vitro, thereby providing insight into the mechanism of BEPD action on VVC.

DNA Damage Signals Repress SYK in Early B Cells to Limit Leukemic Transformation

Early B cell development requires a balance of antagonistic cell programs that drive cellular proliferation as well as maturation. In large pre-B cells, pre-B cell receptor (pre-BCR) signaling activates the ETS family transcription factor, PU.1, and the tyrosine kinase, SYK, which together promote cell proliferation. Subsequently, cells undergo cell cycle arrest and transition to the small pre-B cell stage where immunoglobulin light chain (Igl) gene rearrangement is initiated. Igl gene assembly proceeds through DNA double-stranded breaks (DSBs) generated by the RAG endonuclease. These RAG DSBs induce cellular programs that inhibit pre-BCR signaling through activation of the transcription factor NFkB2. NFkB2 upregulates expression of the SPIC/BCLAF1 transcriptional repressor complex, which displaces PU.1 from the Syk promoter, resulting in reduction of SYK and enforcement of cell cycle arrest. Pediatric pre-B cell leukemia can be driven by errant pre-BCR signaling and increased SYK. We propose that B cell development relies on signals from RAG DSBs to counter pre-BCR signaling and attenuate SYK expression, thereby enforcing a pre-B cell checkpoint that promotes Igl rearrangement and suppresses leukemic transformation. To examine coordination between pre-BCR and the RAG DSB signals in vivo, we developed a novel reporter mouse with a knock-in allele at the endogenous Syk locus encoding an N-terminal fusion of mCherry to SYK (SykmCh). Heterozygous SykmCh/+ mice express equivalent SYK and mCherry-SYK proteins and have normal SYK activity as evidenced by phosphorylation of BLNK, a downstream target of the SYK kinase. To determine if SYK levels are modulated by RAG DSB signals, we generatedRag1-/-:SykmCh/+:IgH:Bcl2 and Art-/-:SykmCh/+:IgH:Bcl2 mice. Rag1-/- mice cannot make DSBs whereas Art-/- mice cannot repair DSBs, due to lack of the DNA damage response protein, Artemis. IgH transgene promotes development to pre-B cell stage and Bcl2 supports survival to permit assessment of DNA damage responses. Flow cytometry revealed lower mCherry-SYK levels in Art-/- small pre-B cells compared to Rag1-/- small pre-B cells, which supports that SYK is suppressed in vivo by RAG DSBs. We next assessed SYK levels during normal B cell development. mCherryhigh and mCherrylow small pre-B cells from SykmCh/+mice were sorted and demonstrated an inverse correlation between SYK and DSB signals. Specifically, relative to mCherryhigh small pre-B cells, mCherrylow small pre-B cells have lower SYK and increased NFkB2 activation, indicating concurrent repressed pre-BCR signals and active RAG DSB signals. Notably, mCherryhigh small pre-B cells have expression of DSB-induced genes Spic, Pim2, and Cd40. Together, these results demonstrate that RAG DSB signals are initiated in SYK-high small pre-B cells and then subsequently promote transition to SYK-low small pre-B cells. To investigate consequences of altered SYK regulation, we generated p53f/f:Bclaf1f/f:Mb1-cre mice, which have selective loss of p53 and Bclaf1 in early B cells. We previously showed that Bclaf1flox/flox:Mb1-cre mice have increased numbers of large pre-B cells with higher Syk expression. However, there were no abnormalities in immature B cell numbers, suggesting additional mechanisms, such as activation of p53 by RAG DSBs, may complement BCLAF1 to enforce B cell development. Indeed, we find that all p53flox/flox:Bclaf1flox/flox:Mb1-cre mice die between 9-12 months of age with malignant expansion of B cells in both spleen and bone marrow. In contrast, no p53flox/flox:Mb1-cre, Bclaf1flox/flox:Mb1-cre, or Bclaf1flox/flox:p53flox/flox mice died or developed abnormal B cells in this time period. The atypical B cells in the p53:Bclaf1 double-deficient mice have altered B220 expression, normal expression of IgM and absence of CD43, consistent with an aberrant immature B cell phenotype. These results demonstrate that RAG DSB signals function to preserve pre-B cell checkpoint and maintain normal B cell maturation. Collectively, these studies establish a novel system for quantitating DNA damage signaling in vivo and for assessing dynamic changes in SYK during B cell maturation. Further, our findings demonstrate a critical function of RAG DSB signals in regulation of early B cell development.

Curcumin alleviates experimental colitis via a potential mechanism involving memory B cells and Bcl-6-Syk-BLNK signaling

Immune dysfunction is the crucial cause in the pathogenesis of inflammatory bowel disease (IBD), which is mainly related to lymphocytes (T or B cells, incl-uding memory B cells), mast cells, activated neutrophils, and macrophages. As the precursor of B cells, the activation of memory B cells can trigger and differentiate B cells to produce a giant variety of inducible B cells and tolerant B cells, whose dysfunction can easily lead to autoimmune diseases, including IBD. To investigate whether or not curcumin (Cur) can alleviate experimental colitis by regulating memory B cells and Bcl-6-Syk-BLNK signaling. Colitis was induced in mice with a dextran sulphate sodium (DSS) solution in drinking water. Colitis mice were given Cur (100 mg/kg/d) orally for 14 con-secutive days. The colonic weight, colonic length, intestinal weight index, occult blood scores, and histological scores of mice were examined to evaluate the curative effect. The levels of memory B cells in peripheral blood of mice were measured by flow cytometry, and IL-1β, IL-6, IL-10, IL-7A, and TNF-α expression in colonic tissue homogenates were analyzed by enzyme-linked immunosorbent assay. Western blot was used to measure the expression of Bcl-6, BLNK, Syk, and other signaling pathway related proteins. After Cur treatment for 14 d, the body weight, colonic weight, colonic length, colonic weight index, and colonic pathological injury of mice with colitis were ameliorated. The secretion of IL-1β, IL-6, TNF-α, and IL-7A was statistically decreased, while the IL-35 and IL-10 levels were considerably increased. Activation of memory B cell subsets in colitis mice was confirmed by a remarkable reduction in the expression of IgM, IgG, IgA, FCRL5, CD103, FasL, PD-1, CD38, and CXCR3 on the surface of CD19+ CD27+ B cells, while the number of CD19+ CD27+ IL-10+ and CD19+ CD27+ Tim-3+ B cells increased significantly. In addition, Cur significantly inhibited the protein levels of Syk, p-Syk, Bcl-6, and CIN85, and increased BLNK and p-BLNK expression in colitis mice. Cur could effectively alleviate DSS-induced colitis in mice by regulating memory B cells and the Bcl-6-Syk-BLNK signaling pathway.

The role of Syk phosphorylation in Fc receptor mediated mIgM+ B lymphocyte phagocytosis in flounder (Paralichthys olivaceus)

Spleen tyrosine kinase (Syk) is a non-receptor protein tyrosine kinase, and it mediates downstream signaling of FcR-mediated immune responses. Our previous work revealed that the expression of Syk was significantly up-regulated in flounder mIgM+ B lymphocytes after phagocytosis of antiserum-opsonized Edwardsiella tarda, which suggested Syk might be involved in Ig-opsonized phagocytosis. In this paper, phospho-Syk (pSyk) inhibitor was used to investigate the potential role of phosphorylated Syk in FcR-mediated phagocytosis of IgM+ B cells. Indirect immunofluorescence assay (IFA) and Western blotting showed that the level of phosphorylated Syk in the mIgM+ B lymphocytes treated with pSyk inhibitor was significantly lower compared to the control group after stimulation with flounder antiserum. Flow cytometry analysis showed that after 3 h incubation with antiserum-opsonized E. tarda, the phagocytosis rates of mIgM+ B lymphocytes from peripheral blood, spleen and head kidney pre-treated with pSyk inhibitor were 48.1%, 40.1% and 43.6% respectively, which were significantly lower than that of the control groups with 58.7%, 53.2% and 57.4%, respectively. And likewise, after pSyk inhibitor treatment, the proportions of mIgM+ B lymphocytes with higher intracellular reactive oxygen species (ROS) levels in peripheral blood, spleen and head kidney decreased to 15.2%, 12.0% and 12.1% from the control level of 26.5%, 25.9% and 26.3%, respectively. Moreover, the expression of three genes affected by pSyk, including phospholipase Cγ1 (PLCγ1), phospholipase Cγ2 (PLCγ2) and phosphatidylinositol 3 kinase (PI3K) were found to be significantly down-regulated in pSyk inhibitor-treated mIgM+ B lymphocytes post phagocytosis. These results suggest that pSyk plays a key role in FcR-mediated phagocytosis and bactericidal activity of mIgM+ B lymphocytes, which promotes further understanding of the regulatory role of pSyk in teleost B cells phagocytosis.

Targeting the splicing isoforms of spleen tyrosine kinase affects the viability of colorectal cancer cells

Spleen tyrosine kinase (Syk) expression have been both positively and negatively associated with tumorigenesis. Our goal was to evaluate the contribution of Syk and its two splice variants, full length Syk (L) and short isoform Syk (S), in the tumor biology of colorectal cancer cells (CRC). The analysis of Syk expression in primary human colorectal tumors, as well as the analysis of TCGA database, revealed a high Syk mRNA expression score in colorectal cancer tumors, suggesting a tumor promotor role of Syk in CRC. Our analysis showed that Syk (L) isoform is highly expressed in the majority of the tumor tissues and that it remains expressed in tumors in which global Syk expression is downregulated, suggesting the dependence of tumors to Syk (L) isoform. We also identified a small cluster of tumor tissues, which express a high proportion of Syk (S) isoform. This specific cluster is associated with overexpressed genes related to translation and mitochondria, and down regulated genes implicated in the progression of mitosis. For our functional studies, we used short hairpin RNA tools to target the expression of Syk in CRC cells bearing the activating K-Ras (G13D) mutation. Our results showed that while global Syk knock down increases cell proliferation and cell motility, Syk (L) expression silencing affects the viability and induces the apoptosis of the cells, confirming the dependence of cells on Syk (L) isoform for their survival. Finally, we report the promising potential of compound C-13, an original non-enzymatic inhibitor of Syk isolated in our group. In vitro studies showed that C-13 exerts cytotoxic effects on Syk-positive CRC cells by inhibiting their proliferation and their motility, and by inducing their apoptosis, while Syk-negative cell lines viability was not affected. Moreover, the oral and intraperitoneal administration of C-13 reduced the tumor growth of CRC DLD-1 cells xenografts in Nude mice in vivo.

Prognostic significance of BLK expression in R-CHOP treated diffuse large B-cell lymphoma

BackgroundThe aim of the present study was to evaluate the prognostic significance of B-cell lymphocyte kinase (BLK) expression for survival outcomes in diffuse large B-cell lymphoma (DLBCL) patients treated with R-CHOP.MethodsWe retrospectively analyzed the medical records of 89 patients from two tertiary referral hospitals. The expression of BLK, SYK, and CDK1 were evaluated in a semi-quantitative method using an H-score, and the proportions of BCL2 and C-MYC were evaluated.ResultsA total of 89 patients received R-CHOP chemotherapy as a first-line chemotherapy. The expression rates of BLK in tumor cells was 39.2% (n = 34). BLK expression status was not significantly associated with clinical variables; however, BLK expression in tumor cells was significantly associated with the expression of both C-MYC and BCL2 (p = .003). With a median follow-up of 60.4 months, patients with BLK expression had significantly lower 5-year progression-free survival (PFS) and overall survival rates (49.8% and 60.9%, respectively) than patients without BLK expression (77.3% and 86.7%, respectively). In multivariate analysis for PFS, BLK positivity was an independent poor prognostic factor (hazard ratio, 2.208; p = .040).ConclusionsHere, we describe the clinicopathological features and survival outcome according to expression of BLK in DLBCL. Approximately 39% of DLBCL patients showed BLK positivity, which was associated as a predictive marker for poor prognosis in patients who received R-CHOP chemotherapy.

SYK Is Associated With Malignant Phenotype and Immune Checkpoints in Diffuse Glioma.

Background: Diffuse glioma, the most common intracranial malignant tumor, is characterized by immunosuppression. The prognostic significance and potential therapeutic value of SYK remain obscure. Here, we explored the performance of SYK in predicting patient outcomes and as a therapeutic target. Methods: The mRNA expression and clinical data for pancancer and normal tissues and more than 2,000 glioma samples were collected from public databases. The expression level of SYK was evaluated by qPCR and IHC. The prognostic value of SYK was assessed using the Kaplan–Meier curves and univariate and multivariate Cox regression analyses. A sequence of immune and stromal infiltration analyses was calculated based on the ESTIMATE algorithm, ssGSEA algorithm, TIMER, and single-cell analysis. The SYK-related subtypes were identified via a Consensus Cluster Plus analysis. Results: SYK was significantly differentially expressed in multiple tumors and normal tissues. Importantly, high-expression SYK was enriched in malignant phenotypes of diffuse gliomas, which was further validated by qPCR and IHC. Survival analysis uncovered that SYK was an independently unfavorable prognostic marker in diffuse glioma. Functional enrichment analysis and immune and stromal infiltration analyses showed that SYK was involved in shaping the immunosuppressive microenvironment of diffuse glioma. Additionally, SYK expression was closely associated with some immune checkpoint molecules and M2 macrophage infiltration, which was validated by IHC and single-cell analysis. Diffuse glioma with Sub1 exhibited a worse prognosis, immunosuppressive microenvironment, and higher expression of immune checkpoint genes. Conclusion: SYK is involved in shaping the immunosuppressive microenvironment and served as a promising prognosis biomarker and immunotherapeutic target for diffuse glioma.

Tumor cell SYK expression modulates the tumor immune microenvironment composition in human cancer via TNF-α dependent signaling

BackgroundThe expression of SYK in cancer cells has been associated with both tumor promoting and tumor suppressive effects. Despite being proposed as anticancer therapeutic target, the possible role of SYK...

Immunomodulatory Effect of Ginsenoside Rb2 Against Cyclophosphamide-Induced Immunosuppression in Mice.

Ginsenoside Rb2 (Rb2), a fundamental saponin produced and isolated from ginseng (Panax ginseng C.A. Meyer), has a wide range of biological actions. The objective of this investigation was to see if ginsenoside Rb2 has any immunomodulatory properties against cyclophosphamide (CTX)-induced immunosuppression. For the positive control group, levamisole hydrochloride (LD) was used. We discovered that intraperitoneal injection of Rb2 (5, 10, 20 mg/kg) could relieve CTX-induced immunosuppression by enhanced immune organ index, reduced the pathological characteristics of immunosuppression, promoted natural killer (NK) cells viability, improved cell-mediated immune response, boosted the IFN-γ (Interferon-gamma), TNF-α (Tumor necrosis factor-alpha), IL-2 (Interleukin-2), and IgG (Immunoglobulin G), as well as macrophage activity like carbon clearance and phagocytic index. Rb2 significantly elevated the mRNA expression of IL-4 (Interleukin-4), SYK (Tyrosine-protein kinase-SYK), IL-2, TNF-α, and IL-6 (Interleukin-6) in the spleen of CTX-injected animals. Molecular docking results showed that Rb2 had excellent binding properties with IL-4, SYK, IL-2, TNF, and IL-6, indicating the target protein might be strongly correlated with the immunomodulatory effect of Rb2. Taken together, ginsenoside Rb2 can improve the immune function that is declined in CTX-induced immunosuppressed mice, the efficacy maybe due to the regulation of related cytokine and mRNA expression.

Pharmacological Inhibition of Spleen Tyrosine Kinase Suppressed Neuroinflammation and Cognitive Dysfunction in LPS-Induced Neurodegeneration Model.

Tyrosine-protein kinase (Syk) plays a potential role in neuroinflammation and adaptive immune responses in several neurodegenerative conditions. Seeing the significant role of Syk in the pathophysiology of neurodegeneration, several pharmacological inhibitors have been developed. One of the known inhibitors of Syk is BAY61-3606, which has shown efficacies in Alzheimer’s disease (AD) through regulating amyloid production. However, little is known about its efficacies in neuroinflammation and neurodegeneration. Our finding showed that Syk expression was up-regulated by lipopolysaccharide (LPS)-dependent manner, and BAY61-3606 significantly suppressed the activated microglia (ionized calcium-binding adaptor molecule 1 [Iba-1]) and the inflammatory cytokines (tumor necrosis factor-alpha [TNF-α], interleukin 1-beta [IL-1β], IL-6) and other inflammatory mediators (nuclear factor kappa B [NF-κB], cyclooxygenase-2 [Cox-2], and inducible nitric axide synthase [iNOS]) in the lipopolysaccharide (LPS)-treated in vivo and in vitro models. Moreover, BAY61-3606 significantly reduced microglia-mediated neuronal cell death by regulating the expression of Cytochrome C and Bim (B-cell lymphoma 2 [BCL-2] interacting mediator of cell death) in the LPS-treated mice brain and HT22 cells. Furthermore, the expression of synaptic markers, synaptosomal-associated protein, 25 kDa (SNAP25), synaptophysin (Syp), and postsynaptic density protein-95 (PSD95) in LPS-challenged mice showed that BAY61-3606 significantly recovered the synaptic markers. Finally, we have analyzed the effects of BAY61-3606 against memory and cognitive dysfunctions in the LPS injected mice. The Y-maze test and Passive avoidance test suggested that BAY61-3606 significantly protected against LPS-induced cognitive and memory dysfunctions. The current findings not only highlight the mechanisms of Syk in the pathophysiology of neuro-inflammation, but also support the therapeutic efficacy of BAY61-3606 in the management of neurodegeneration.

Butyl alcohol extract of Baitouweng Decoction alleviates vulvovaginal candidiasis in mice by downregulating NLRP3 inflammasome and related signal pathways

This study aims to explore the effect of butyl alcohol extract of Baitouweng Decoction(BAEB) on vulvovaginal candidiasis(VVC) in mice and to clarify the mechanism from Toll-like receptors(TLRs)/MyD88 and Dectin-1/Syk signal pathways and NLRP3 inflammasome. To be specific, female KM mice were randomized into control group(i.g., normal saline), model group, fluco-nazole group(i.g., 20 mg·kg~(-1)), and low-dose, medium-dose, and high-dose BAEB groups(i.g., 20, 40, and 80 mg·kg~(-1), respectively). VVC was induced in mice except the control group. After the modeling, administration began and lasted 7 days. The ge-neral conditions and body weight of mice were recorded every day. On the 1 st, 3 rd, 7 th, and 14 th after vaginal infection by Candida albicans, the fungal load in the vaginal lavage fluid of the mice was measured with the plate method, and the morphology of C. albicans in vaginal lavage fluid was observed based on Gram staining. After the mice were killed, vaginal tissues were subjected to hematoxylin-eosin(HE) staining and periodic acid-Schiff(PAS) staining for vaginal histopathological analysis. The content of cytokines in vaginal lavage fluid, such as interleukin(IL)-1β, IL-18, tumor necrosis factor-α(TNF-α), IL-6, and S100 a8, was determined by enzyme-linked immunosorbent assay(ELISA), and content of reactive oxygen species(ROS) in vaginal tissues by tissue ROS detection kit. The protein expression of NLRP3, ASC, caspase-1, Dectin-1, Syk, MyD88, TLR2, TLR4, and nuclear factor-κB(NF-κB) in vaginal tissues was detected by Western blot, and the levels and distribution of NLRP3, Dectin-1, Syk, MyD88, TLR2, and TLR4 in vaginal tissues were determined with the immunohistochemical method. The results show that BAEB can improve the general conditions of VVC mice, reduce the fungal load and C. albicans hyphae in vaginal secretion, decrease ROS content in vaginal tissues and content of cytokines in vaginal lavage fluid, and down-regulate the expression of NLRP3, ASC, caspase-1, Dectin-1, Syk, MyD88, TLR2, TLR4, and NF-κB in vaginal tissues. The above results indicate that BAEB exerts therapeutic effect on VVC mice by down-regulating the key proteins in the TLRs/MyD88 and Dectin-1/Syk signal pathways and NLRP3 inflammasome.

Perilla Leaf Extract (PLE) Attenuates COPD Airway Inflammation via the TLR4/Syk/PKC/NF-κB Pathway In Vivo and In Vitro.

Chronic obstructive pulmonary disease (COPD) is a complex and heterogeneous disease characterized by persistent airflow limitation but still lacking effective treatments. Perilla frutescens (L.) Britt., an important traditional medicinal plant with excellent antioxidant and anti-inflammatory properties, is widely used for the treatment of respiratory disease in China. However, its protective activity and mechanism against COPD airway inflammation have not been fully studied. Here, the anti-inflammatory effects of the PLE were investigated, and its underlying mechanisms were then elucidated. The presented results suggested a notable effect of the PLE on airway inflammation of COPD, by significantly ameliorating inflammatory cell infiltration in lung tissue, lessening leukocytes (lymphocytes, neutrophils, and macrophages) and inflammatory mediators (interleukin 4 (IL-4), IL-6, IL-17A, interferon γ (IFN-γ), and tumor necrosis factor α (TNF-α)) in the bronchoalveolar lavage fluid (BALF) of cigarette smoke (CS)/lipopolysaccharide (LPS)-induced COPD mice in vivo and inhibiting the production of inflammatory factors (nitric oxide (NO), IL-6, and TNF-α) and intracellular reactive oxygen species (ROS) in LPS-stimulated RAW264.7 cells in vitro. For further extent, PLE treatment significantly suppressed the expression and phosphorylation of TLR4, Syk, PKC, and NF-κB p65 in vivo and their mRNA in vitro. Subsequently, by co-treating with their inhibitors in vitro, its potential mechanism via TLR4/Syk/PKC/NF-κB p65 signals was disclosed. In summary, the obtained results indicated a noteworthy effective activity of the PLE on COPD inflammation, and partly, the TLR4/Syk/PKC/NF-κB p65 axis might be the potential mechanism.

Bob.1 Blocks a SYK Protein Degradation Pathway in Mice That Develop Chronic Graft Versus Host Disease Manifestations

After elucidating a role for aberrant B Cell Receptor (BCR) signaling in chronic graft versus host disease (cGVHD) (Allen et al, Blood. 2014;123:2108), we showed that the proximal BCR molecule SYK is a viable therapeutic target in mice and patients (Flynn et al, Blood 2015; 125:4085. Poe et al, JCI Insight. 2018;3:e122430. NCT02611063). Increased SYK protein levels associate with enhance phosphorylation of SYK activation sites, leading to immediate BCR responsiveness in cGVHD. In mice that develop cGVHD manifestations after allogeneic bone marrow transplantation (BMT), BCR responsiveness is promoted by B Cell Activating Factor (BAFF) (Jia et al, Blood. 2021;137:2544). Instead of SYK protein levels decreasing upon engagement with surrogate antigen to regulate excessive BCR-signaling, SYK protein was maintained when BAFF and alloantigen were present. In the present study, we address a potential molecular mechanism underpinning SYK protein maintenance in cGVHD B cells.In an unbiased scRNAseq study examining purified B cells from active cGVHD patients, we found that Pou2af1 was significantly altered within a BCR-activated B cell subset (Poe et al, Blood. 2019; 134; Suppl1:874-874). Pou2af1 encodes B cell Oct binding factor 1 (Bob.1), a prime candidate for further study. Bob.1 is a transcriptional coactivator that is also able to bind to SYK and regulate SYK protein stability (Siegel et al, Cell. 2006; 125:761). To determine whether BAFF promoted increased Bob.1 expression we examined B cells from BAFF transgenic (Tg) mice. Consistent with a role for this molecule in blocking SYK degradation, Bob.1 protein levels were notably higher in BAFF-Tg B cells. Intracellular flow cytometry analyses of B cells at day 40 after BMT (cGVHD n=9 vs control n=5 mice) confirmed that Bob.1 is significantly higher in cGVHD mouse B cells (p=0.0002, data not shown). We then investigated whether phosphorylation of a SYK negative regulatory site after BCR-activation was affected by BAFF. Since tyrosine 317 (SYK-Y317) phosphorylation is known to initiate ubiquitination and degradation of SYK, we stimulated purified B cells through the BCR before preparing cell lysates and performing Western blot analyses to measure SYK-Y317 phosphorylation, total SYK, LYN, Bob.1 and BLNK. As shown in a representative blot in Fig 1A, phosphorylation of SYK-Y317 (pSYK-Y317) was significantly decreased in B cells from BAFF transgenic (BAFF-Tg) mice compared to those from wild type (WT) mice. These data suggest that BAFF promotes SYK through Bob.1.Hypothesizing that Bob.1 is required to maintain SYK protein after allogeneic BMT, we employed Bob.1 knockout mice. After verifying that heterozygous mice (Bob.1 +/-) had lower Bob.1 protein levels and comparable B cell numbers to WT mice, we investigated the effect of Bob.1 reduction on SYK levels. Bob.1 +/- mice were crossed with BAFF-Tg mice and then SYK expression in B cells was determined using intracellular flow cytometry. We found that SYK protein levels were significantly increased in B cells exposed to a high BAFF environment and that SYK levels were significantly lower when Bob.1 protein was decreased (Fig 1B). These data demonstrate that Bob.1 is needed in vivo to maintain BAFF-mediated SYK protein maintenance after BCR engagement. Finally, we evaluated whether Bob.1 affected phosphorylation at SYK-Y317 in B cells from cGVHD mice. Splenic B cells were purified 41 days after BMT from recipients of B6 syngeneic (Syn), BM alone (BM only, control) or BM plus splenocytes (BM+Sp, cGVHD) and then stimulated with anti-IgM for 30 seconds before pSyk-Y317 and total SYK were detected by Western blot (Fig 1C). Using Image Studio Lite software, we normalized the intensity of pSyk-Y317 to actin in n=4 (Syn-BM+Sp), n=5 (BM only) and n=4 (BM+Sp) and found a significant decrease in pSyk-Y317 in cGVHD mice. We are currently investigating potential pathologic effects of Bob.1 after BMT in our mouse model and in patient cells.In summary, we have now demonstrated that, Bob.1 is required to maintain SYK protein in B cells after allogeneic BMT in mice that develop cGVHD. Our data suggest that Bob.1 maintains the stability of BCR proximal SYK by blocking a known SYK degradation pathway (Fig 1D). Improving our understanding of Bob.1 and SYK protein homeostasis after HCT will impact how we implement SYK inhibitors in patients.This work was supported the National Institutes of Health (NHLBI R01 HL 129061-06). [Display omitted] DisclosuresSarantopoulos: Rigel: Other: Advisory Board.

Effects of G-Rh2 on mast cell-mediated anaphylaxis via AKT-Nrf2/NF-κB and MAPK-Nrf2/NF-κB pathways

BackgroundThe effect of ginsenoside Rh2 (G-Rh2) on mast cell-mediated anaphylaxis remains unclear. Herein, we investigated the effects of G-Rh2 on OVA-induced asthmatic mice and on mast cell-mediated anaphylaxis. MethodsAsthma model was established for evaluating airway changes and ear allergy. RPMCs and RBL-2H3 were used for in vitro experiments. Calcium uptake, histamine release and degranulation were detected. ELISA and Western blot measured cytokine and protein levels, respectively. ResultsG-Rh2 inhibited OVA-induced airway remodeling, the production of TNF-α, IL-4, IL-8, IL-1β and the degranulation of mast cells of asthmatic mice. G-Rh2 inhibited the activation of Syk and Lyn in lung tissue of OVA-induced asthmatic mice. G-Rh2 inhibited serum IgE production in OVA induced asthmatic mice. Furthermore, G-Rh2 reduced the ear allergy in IgE-sensitized mice. G-Rh2 decreased the ear thickness. In vitro experiments G-Rh2 significantly reduced calcium uptake and inhibited histamine release and degranulation in RPMCs. In addition, G-Rh2 reduced the production of IL-1β, TNF-α, IL-8, and IL-4 in IgE-sensitized RBL-2H3 cells. Interestingly, G-Rh2 was involved in the FcεRI pathway activation of mast cells and the transduction of the Lyn/Syk signaling pathway. G-Rh2 inhibited PI3K activity in a dose-dependent manner. By blocking the antigen-induced phosphorylation of Lyn, Syk, LAT, PLCγ2, PI3K ERK1/2 and Raf-1 expression, G-Rh2 inhibited the NF-κB, AKT-Nrf2, and p38MAPK-Nrf2 pathways. However, G-Rh2 up-regulated Keap-1 expression. Meanwhile, G-Rh2 reduced the levels of p-AKT, p38MAPK and Nrf2 in RBL-2H3 sensitized IgE cells and inhibited NF-κB signaling pathway activation by activating the AKT-Nrf2 and p38MAPK-Nrf2 pathways. ConclusionG-Rh2 inhibits mast cell-induced allergic inflammation, which might be mediated by the AKT-Nrf2/NF-κB and p38MAPK-Nrf2/NF-κB signaling pathways.

Analysis of SYK Gene as a Prognostic Biomarker and Suggested Potential Bioactive Phytochemicals as an Alternative Therapeutic Option for Colorectal Cancer: An In-Silico Pharmaco-Informatics Investigation.

Background: SYK gene regulates the expression of SYK kinase (Spleen tyrosine kinase), an important non-receptor protein-tyrosine kinase for immunological receptor-mediated signaling, which is also considered a tumor growth metastasis initiator. An onco-informatics analysis was adopted to evaluate the expression and prognostic value of the SYK gene in colorectal cancer (CRC), the third most fatal cancer type; of late, it may be a biomarker as another targeted site for CRC. In addition, identify the potential phytochemicals that may inhibit the overexpression of the SYK kinase protein and minimize the human CRC. Materials & Methods: The differential expression of the SYK gene was analyzed using several transcriptomic databases, including Oncomine, UALCAN, GENT2, and GEPIA2. The server cBioPortal was used to analyze the mutations and copy number alterations, whereas GENT2, Gene Expression Profiling Interactive Analysis (GEPIA), Onco-Lnc, and PrognoScan were used to examine the survival rate. The protein-protein interaction network of SYK kinase and its co-expressed genes was conducted via Gene-MANIA. Considering the SYK kinase may be the targeted site, the selected phytochemicals were assessed by molecular docking using PyRx 0.8 packages. Molecular interactions were also observed by following the Ligplot+ version 2.2. YASARA molecular dynamics simulator was applied for the post-validation of the selected phytochemicals. Results: Our result reveals an increased level of mRNA expression of the SYK gene in colorectal adenocarcinoma (COAD) samples compared to those in normal tissues. A significant methylation level and various genetic alterations recurrence of the SYK gene were analyzed where the fluctuation of the SYK alteration frequency was detected across different CRC studies. As a result, a lower level of SYK expression was related to higher chances of survival. This was evidenced by multiple bioinformatics platforms and web resources, which demonstrated that the SYK gene can be a potential biomarker for CRC. In this study, aromatic phytochemicals, such as kaempferol and glabridin that target the macromolecule (SYK kinase), showed higher stability than the controls, and we have estimated that these bioactive potential phytochemicals might be a useful option for CRC patients after the clinical trial. Conclusions: Our onco-informatics investigation suggests that the SYK gene can be a potential prognostic biomarker of CRC. On the contrary, SYK kinase would be a major target, and all selected compounds were validated against the protein using in-silico drug design approaches. Here, more in vitro and in vivo analysis is required for targeting SYK protein in CRC.

Effect of piceatannol against malignant melanoma in vivo and in vitro.

To study the antitumor effect of piceatannol (PIC) on malignant melanoma in vitro and in vivo. B16F10 cells were cultured in vitro and treated with gradient concentrations of PIC. Cell viability was detected with methyl thiazolyl tetrazolium (MTT) assay; matrix metalloproteinase (MMP)-2, MMP-9, vascular endothelial growth factor (VEGF), spleen tyrosine kinase (Syk), and p-Syk were detected with Western blot; migration ability was detected with wound healing assay; invasion ability was detected with Transwell assay. Syk expression was suppressed through RNA interference for the detection of the possible mechanism of PIC in melanoma. An in vivo study was established by creating B16F10-bearing mice with intraperitoneal injection of PIC. The cell viability of B16F10 decreased with increasing PIC concentration. The results of the Transwell assay showed that invasion ability decreased with increasing PIC concentration, and healing time was prolonged at increased PIC concentration in the wound healing assay. Western blot results showed that PIC mainly inhibited the phosphorylation of Syk and inhibited the expression of MMP-2, MMP-9, and VEGF. RNA interference pointed out that blocking the expression of Syk can reveal the same inhibition effect on B16F10 cells as PIC. In vivo study revealed that different concentrations of PIC cangreatly inhibit melanoma progression. PIC might block the progression of malignant melanoma by inhibiting spleen tyrosine kinase.

MiR-96-5p is the tumor suppressor in osteosarcoma via targeting SYK

BackgroundOsteosarcoma is a multiple malignant tumor in adolescents. MicroRNAs (MiRNAs) have been found to express abnormally in OS tissues and are considered as potential targets for OS prognosis and treatment. MethodsMiR-96-5p and SYK expression in clinical samples, osteoblast and OS cell lines were detected. The changes of cell proliferation, apoptosis, adhesion and metastasis of OS cells were detected by CCK-8, BrdU, caspase-3 activity and transwell assay. Dual luciferase report analysis and RNA pull-down were used to confirm binding relation of miR-96-5p and SYK. ResultsMiR-96-5p was increased in OS tissue and cells. Moreover, miR-96-5p inhibits proliferation, adhesion and migration of HOS and Saos-2 cells, and promotes cell apoptosis. SYK has been identified to be targeted by miR-96-5p. Overexpressed SYK inhibits the suppressive impact of miR-96-5 on OS cells. ConclusionMiR-96-5p may function as an effective target in OS treatment.

Complement activation and increased expression of Syk, mucin-1 and CaMK4 in kidneys of patients with COVID-19

Acute and chronic kidney failure is common in hospitalized patients with COVID-19, yet the mechanism of injury and predisposing factors remain poorly understood. We investigated the role of complement activation by determining the levels of deposited complement components (C1q, C3, FH, C5b-9) and immunoglobulin along with the expression levels of the injury-associated molecules spleen tyrosine kinase (Syk), mucin-1 (MUC1) and calcium/calmodulin-dependent protein kinase IV (CaMK4) in the kidney tissues of people who succumbed to COVID-19. We report increased deposition of C1q, C3, C5b-9, total immunoglobulin, and high expression levels of Syk, MUC1 and CaMK4 in the kidneys of COVID-19 patients. Our study provides strong rationale for the expansion of trials involving the use of inhibitors of these molecules, in particular C1q, C3, Syk, MUC1 and CaMK4 to treat patients with COVID-19.

Chronic T cell receptor stimulation unmasks NK receptor signaling in peripheral T cell lymphomas via epigenetic reprogramming.

Peripheral T cell lymphomas (PTCLs) represent a significant unmet medical need with dismal clinical outcomes. The T cell receptor (TCR) is emerging as a key driver of T lymphocyte transformation. However, the role of chronic TCR activation in lymphomagenesis and in lymphoma cell survival is still poorly understood. Using a mouse model, we report that chronic TCR stimulation drove T cell lymphomagenesis, whereas TCR signaling did not contribute to PTCL survival. The combination of kinome, transcriptome, and epigenome analyses of mouse PTCLs revealed a NK cell-like reprogramming of PTCL cells with expression of NK receptors (NKRs) and downstream signaling molecules such as Tyrobp and SYK. Activating NKRs were functional in PTCLs and dependent on SYK activity. In vivo blockade of NKR signaling prolonged mouse survival, demonstrating the addiction of PTCLs to NKRs and downstream SYK/mTOR activity for their survival. We studied a large collection of human primary samples and identified several PTCLs recapitulating the phenotype described in this model by their expression of SYK and the NKR, suggesting a similar mechanism of lymphomagenesis and establishing a rationale for clinical studies targeting such molecules.