Myeloid Differentiation Primary Response Gene Research Articles

Subtype-specific addiction of the activated B-cell subset of diffuse large B-cell lymphoma to FOXP1

High expression of the forkhead box P1 (FOXP1) transcription factor distinguishes the aggressive activated B cell (ABC) diffuse large B-cell lymphoma (DLBCL) subtype from the better prognosis germinal center B-cell (GCB)-DLBCL subtype and is highly correlated with poor outcomes. A genetic or functional role for FOXP1 in lymphomagenesis, however, remains unknown. Here, we report that sustained FOXP1 expression is vital for ABC-DLBCL cell-line survival. Genome-wide analyses revealed direct and indirect FOXP1 transcriptional enforcement of ABC-DLBCL hallmarks, including the classical NF-κB and MYD88 (myeloid differentiation primary response gene 88) pathways. FOXP1 promoted gene expression underlying transition of the GCB cell to the plasmablast--the transient B-cell stage targeted in ABC-DLBCL transformation--by antagonizing pathways distinctive of GCB-DLBCL, including that of the GCB "master regulator," BCL6 (B-cell lymphoma 6). Cell-line derived FOXP1 target genes that were highly correlated with FOXP1 expression in primary DLBCL accurately segregated the corresponding clinical subtypes of a large cohort of primary DLBCL isolates and identified conserved pathways associated with ABC-DLBCL pathology.

Grancalcin (GCA) modulates Toll‐like receptor 9 (TLR9) mediated signaling through its direct interaction with TLR9

Toll-like receptors (TLRs) are playing important roles in stimulating the innate immune response and intensifying adaptive immune response against invading pathogens. Appropriate regulation of TLR activation is important to maintain a balance between preventing tumor activation and inhibiting autoimmunity. Toll-like receptor 9 (TLR9) senses microbial DNA in the endosomes of plasmacytoid dendritic cells and triggers myeloid differentiation primary response gene 88 (MyD88) dependent nuclear factor kappa B (NF-κB) pathways and type I interferon (IFN) responses. However, mechanisms of how TLR9 signals are mediated and which molecules are involved in controlling TLR9 functions remain poorly understood. Here, we report that penta EF-hand protein grancalcin (GCA) interacts and binds with TLR9 in a yeast two-hybrid system and an overexpression system. Using siRNA-mediated knockdown experiments, we also revealed that GCA positively regulates type I IFN production, cytokine/chemokine production through nuclear localization of interferon regulatory factor 7 (IRF7), NF-κB activation, and mitogen-activated protein kinase (MAPK) activation in plasmacytoid dendritic cells. Our results indicate that heterodimerization of GCA and TLR9 is important for TLR9-mediated downstream signaling and might serve to fine tune processes against viral infection.

In vitro antioxidative and anti-inflammatory effects of the compound K-rich fraction BIOGF1K, prepared from Panax ginseng

BackgroundBIOGF1K, a compound K-rich fraction prepared from the root of Panax ginseng, is widely used for cosmetic purposes in Korea. We investigated the functional mechanisms of the anti-inflammatory and antioxidative activities of BIOGF1K by discovering target enzymes through various molecular studies. MethodsWe explored the inhibitory mechanisms of BIOGF1K using lipopolysaccharide-mediated inflammatory responses, reporter gene assays involving overexpression of toll-like receptor adaptor molecules, and immunoblotting analysis. We used the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay to measure the antioxidative activity. We cotransfected adaptor molecules, including the myeloid differentiation primary response gene 88 (MyD88) and Toll/interleukin-receptor domain containing adaptor molecule-inducing interferon-β (TRIF), to measure the activation of nuclear factor (NF)-κB and interferon regulatory factor 3 (IRF3). ResultsBIOGF1K suppressed lipopolysaccharide-triggered NO release in macrophages as well as DPPH-induced electron-donating activity. It also blocked lipopolysaccharide-induced mRNA levels of interferon-β and inducible nitric oxide synthase. Moreover, BIOGF1K diminished the translocation and activation of IRF3 and NF-κB (p50 and p65). This extract inhibited the upregulation of NF-κB-linked luciferase activity provoked by phorbal-12-myristate-13 acetate as well as MyD88, TRIF, and inhibitor of κB (IκBα) kinase (IKKβ), and IRF3-mediated luciferase activity induced by TRIF and TANK-binding kinase 1 (TBK1). Finally, BIOGF1K downregulated the NF-κB pathway by blocking IKKβ and the IRF3 pathway by inhibiting TBK1, according to reporter gene assays, immunoblotting analysis, and an AKT/IKKβ/TBK1 overexpression strategy. ConclusionOverall, our data suggest that the suppression of IKKβ and TBK1, which mediate transcriptional regulation of NF-κB and IRF3, respectively, may contribute to the broad-spectrum inhibitory activity of BIOGF1K.

Homoplantaginin Inhibits Palmitic Acid-induced Endothelial Cells Inflammation by Suppressing TLR4 and NLRP3 Inflammasome.

Palmitic acid (PA)-induced vascular endothelial inflammation plays a pivotal role in the occurrence and development of vascular diseases. The present study was conducted to examine the effect of homoplantaginin, a main flavonoid from a traditional Chinese medicine Salvia plebeia R. Br., on PA-treated human umbilical vein endothelial cells inflammation and the underlying molecular mechanism. Firstly, we found that homoplantaginin (0.1, 1, 10 μM) dose-dependently reduced expression of toll-like receptor-4 evoked by PA (100 μM). The inhibitory effect of homoplantaginin was further confirmed under lipopolysaccharide challenge. In addition, downstream adapted proteins including myeloid differentiation primary response gene 88, toll/interleukin-1 receptor-domain containing adaptor-inducing interferon-β and tumor necrosis factors receptor associated factor-6 were successfully inhibited by homoplantaginin under PA treatment. Also, we found that homoplantaginin tightly controlled PA-induced reactive oxygen species to prevent nucleotide-binding domain-like receptor 3 (NLRP3) inflammasome activation by suppressing reactive oxygen species-sensitive thioredoxin-interacting protein, NLRP3, and caspase-1. Meanwhile, protein and mRNA levels of inflammatory mediators (interleukin-1β, intercellular cell adhesion molecule-1, and monocyte chemotactic protein-1) were decreased by homoplantaginin. Furthermore, homoplantaginin restored PA-impaired nitric oxide generation. Taken together, these results indicated that homoplantaginin protected endothelial cells from ameliorating PA-induced endothelial inflammation via suppressing toll-like receptor-4 and NLRP3 pathways, and restoring nitric oxide generation, suggesting it may be a potential candidate for further development in the prevention and treatment of vascular diseases.

Clinical Significance of Toll-Like Receptor and Toll-Like Receptor Blocker

The mammalian Toll-like receptor (TLR) family, consisting of 13 members, plays an important role in innate recognition of specific patterns of microbial products. TLR-dependent recognition subsequently causes an activation of antigen-specific adaptive immunity. TLR-mediated signaling pathways consist of two pathways that induce gene expression: the myeloid differentiation primary response gene 88 (MyD88)-dependent pathway and Toll/interleukin-1 receptor-domain containing adaptor protein-inducing interferon--dependent pathway. Synthetic TLR agonists, as well as TLR antagonists, affect and manipulate the host defense systems, and some of these immunomodulating agents may help to overcome intrinsic disturbances of the TLR system to offer new treatment options in urinary tract infection (UTI). Future studies are necessary to clarify additional associations between TLRs and severity of UTI, which may help in developing new treatment options.

Inhibition of TLR4 Signalling-Induced Inflammation Attenuates Secondary Injury after Diffuse Axonal Injury in Rats

Increasing evidence suggests that secondary injury after diffuse axonal injury (DAI) damages more axons than the initial insult, but the underlying mechanisms of this phenomenon are not fully understood. Recent studies show that toll-like receptor 4 (TLR4) plays a critical role in promoting adaptive immune responses and have been shown to be associated with brain damage. The purpose of this study was to investigate the role of the TLR4 signalling pathway in secondary axonal injury in the cortices of DAI rats. TLR4 was mainly localized in microglial cells and neurons, and the levels of TLR4 downstream signalling molecules, including TLR4, myeloid differentiation primary response gene 88, toll/IR-1-(TIR-) domain-containing adaptor protein inducing interferon-beta, interferon regulatory factor 3, interferon β, nuclear factor κB (NF-κB) p65, and phospho-NF-κB p65, significantly increased and peaked at 1 d after DAI. Inhibition of TLR4 by TAK-242 attenuated apoptosis, neuronal and axonal injury, and glial responses. The neuroprotective effects of TLR4 inhibition were associated with decreases in the levels of TLR4 downstream signalling molecules and inflammatory factors, including interleukin-1β, interleukin-6, and tumour necrosis factor-α. These results suggest that the TLR4 signalling pathway plays an important role in secondary injury and may be an important therapeutic target following DAI.

A Multifaceted Role for Myd88-Dependent Signaling in Progression of Murine Mammary Carcinoma.

Previous data obtained in our laboratory suggested that there may be constitutive signaling through the myeloid differentiation primary response gene 88 (Myd88)-dependent signaling cascade in murine mammary carcinoma. Here, we extended these findings by showing that, in the absence of an added Toll-like receptor (TLR) agonist, the myddosome complex was preformed in 4T1 tumor cells, and that Myd88 influenced cytoplasmic extracellular signal–regulated kinase (Erk)1/Erk2 levels, nuclear levels of nuclear factor-kappaB (NFκB) and signal transducer and activator of transcription 5 (STAT5), tumor-derived chemokine (C–C motif) ligand 2 (CCL2) expression, and in vitro and in vivo tumor growth. In addition, RNA-sequencing revealed that Myd88-dependent signaling enhanced the expression of genes that could contribute to breast cancer progression and genes previously associated with poor outcome for patients with breast cancer, in addition to suppressing the expression of genes capable of inhibiting breast cancer progression. Yet, Myd88-dependent signaling in tumor cells also suppressed expression of genes that could contribute to tumor progression. Collectively, these data revealed a multifaceted role for Myd88-dependent signaling in murine mammary carcinoma.

Cigarette smoke-induced necroptosis and DAMP release trigger neutrophilic airway inflammation in mice.

Recent data indicate a role for airway epithelial necroptosis, a regulated form of necrosis, and the associated release of damage-associated molecular patterns (DAMPs) in the development of chronic obstructive pulmonary disease (COPD). DAMPs can activate pattern recognition receptors (PRRs), triggering innate immune responses. We hypothesized that cigarette smoke (CS)-induced epithelial necroptosis and DAMP release initiate airway inflammation in COPD. Human bronchial epithelial BEAS-2B cells were exposed to cigarette smoke extract (CSE), and necrotic cell death (membrane integrity by propidium iodide staining) and DAMP release (i.e., double-stranded DNA, high-mobility group box 1, heat shock protein 70, mitochondrial DNA, ATP) were analyzed. Subsequently, BEAS-2B cells were exposed to DAMP-containing supernatant of CS-induced necrotic cells, and the release of proinflammatory mediators [C-X-C motif ligand 8 (CXCL-8), IL-6] was evaluated. Furthermore, mice were exposed to CS in the presence and absence of the necroptosis inhibitor necrostatin-1, and levels of DAMPs and inflammatory cell numbers were determined in bronchoalveolar lavage fluid. CSE induced a significant increase in the percentage of necrotic cells and DAMP release in BEAS-2B cells. Stimulation of BEAS-2B cells with supernatant of CS-induced necrotic cells induced a significant increase in the release of CXCL8 and IL-6, in a myeloid differentiation primary response gene 88-dependent fashion. In mice, exposure of CS increased the levels of DAMPs and numbers of neutrophils in bronchoalveolar lavage fluid, which was statistically reduced upon treatment with necrostatin-1. Together, we showed that CS exposure induces necrosis of bronchial epithelial cells and subsequent DAMP release in vitro, inducing the production of proinflammatory cytokines. In vivo, CS exposure induces neutrophilic airway inflammation that is sensitive to necroptosis inhibition.

MicroRNA-155 silencing enhances inflammatory response and lipid uptake in oxidized low-density lipoprotein-stimulated human THP-1 macrophages.

It has been proposed that the inflammatory response of monocytes/macrophages induced by oxidized low-density lipoprotein (oxLDL) is a key event in the pathogenesis of atherosclerosis. MicroRNA-155 (miR-155) is an important regulator of the immune system and has been shown to be involved in acute inflammatory response. However, the function of miR-155 in oxLDL-stimulated inflammation and atherosclerosis remains unclear. Here, we show that the exposure of human THP-1 macrophages to oxLDL led to a marked up-regulation of miR-155 in a dose-dependent manner. Silencing of endogenous miR-155 in THP-1 cells using locked nucleic acid-modified antisense oligonucleotides significantly enhanced oxLDL-induced lipid uptake, up-regulated the expression of scavenger receptors (lectinlike oxidized LDL receptor-1, cluster of differentiation 36 [CD36], and CD68), and promoted the release of several cytokines including interleukin (IL)-6, -8, and tumor necrosis factor α (TNF-α). Luciferase reporter assay showed that targeting miR-155 promoted nuclear factor-kappa B (NF-κB) nuclear translocation and potentiated the NF-κB-driven transcription activity. Moreover, miR-155 knockdown resulted in a marked increase in the protein amount of myeloid differentiation primary response gene 88 (MyD88), an important adapter protein used by Toll-like receptors to activate the NF-κB pathway. Our data demonstrate that miR-155 serves as a negative feedback regulator in oxLDL-stimulated THP-1 inflammatory responses and lipid uptake and thus might have potential therapeutic implications in atherosclerosis.

Insulin inhibits inflammation and promotes atherosclerotic plaque stability via PI3K-Akt pathway activation

Toll-like receptor (TLR) 4 induced inflammation was reported to play an important role in atherosclerotic plaque stability. Recent studies indicated that insulin could inhibit inflammation by activating phosphatidylinositol 3-kinase-Akt-dependent (PI3K-Akt) signaling pathway. In the current study, we hypothesized that insulin would inhibit TLR4 induced inflammation via promoting PI3K-Akt activation, thus enhancing the stabilization of atherosclerotic plaques. In order to mimic the process of plaque formation, monocyte-macrophage lineage RAW264.7 were cultured and induced to form foam cells by oxidized LDL (ox-LDL). Oil red O staining results showed that insulin significantly restrained ox-LDL-induced foam cell formation. Analysis of inflammatory reaction during foam cell formation indicated that insulin significantly down-regulated the expression of tumor necrosis factor (TNF)-α, interleukin (IL)-6 levels, inhibited TLR4, myeloid differentiation primary response gene (MyD) 88 and nuclear factor (NF)-κB. Further mechanism analysis showed that pretreating with the PI3K blocker, wortmannin dramatically dampened the insulin-induced up-regulation of pAkt expression. Additionally, blockade of PI3K-Akt signaling also dampened the immunosuppression effect brought by insulin. Following the construction of a rodent atherosclerosis model, pretreatment of insulin resulted in an evident decrease in lipid deposition of the blood vessel wall, serum levels of TNF-α and IL-6, and numbers of infiltrated macrophages and foam cells. Taken together, these results suggested that insulin might inhibit inflammation and promote atherosclerotic plaque stability via the PI3K-Akt pathway by targeting TLR4-MyD88-NF-κB signaling. Our findings may provide a potential target for the prevention of cardiovascular disease.

MYd88 Blockade Caused Reduction of PDL1 on Tumor Infiltrating MDSCs in a Murine Model of Melanoma

Introduction: Myeloid differentiation primary response gene 88 (Myd 88) is an important adaptor molecule for the activation of NADPH oxidase and regulation of arginase-1, which are responsible for the suppressive function of myeloid-derived suppressor cells (MDSCs). Blockade of Myd88 signaling induces antitumor effects in mice by skewing the immunosuppressive function of myeloid-derived suppressor cells. As the PD-L1/PD-1 axis has been characterized as a potent inhibitor of immune activation, particularly through inhibition of effector T cell function, we characterized the effect of Myd88 on checkpoint expression on tumor-infiltrating MDSC/T cells in a murine model melanoma.Methods: Pathogen-free 8-10-week-old WT(B6-background) and Myd 88−/− mice that been backcrossed to a C57BL/6 genetic background were challenged with 1 × 106 B16 (F1) tumor cells s.c. On day 14, mice were sacrificed and spleen and tumors were removed and digested into single-cell suspensions, blocked with anti-FcR mAbs and analyzed for surface and intracellular staining by flow cytometry. We analyzed CD11b+/Gr-1+hi/int myeloid cells subsets and T cells in the blood, spleen and tumors of mice by flow cytomery.Results: The growth of B16 melanoma tumor was significantly slower in Myd 88−/− mice compared with WT mice. No significant difference between two groups was found in the frequency of absolute number of MDSC subsets and expression of PDL1 check-point marker on spleen-derived MDSC subsets. In contrast CD4(+) and C8(+) T cells residing in spleens of Myd88(-/-) mice showed increased expression of TNF-α/IFN-α and GrZB compared with T cells from wild-type mice following short-term activation with PMA/iono. Of note, the frequencies and absolute numbers of infiltrating CD11b+/Gr1+ MDSC in tumor-bearing Myd 88−/− mice were lower than those in WT mice. Also we found that viable CD11b+/Gr1+ MDSC subsets from WT mice expressed higher level of PD-L1 compared with MDSCs from Myd 88−/− mice in concordance with the reduced expression of PD-1 on tumor infiltrating CD4+ T cells in Myd 88−/− mice. Collectively, the profile of PD-L1 and PD-1 expression in tumor microenvironments is favorably altered to enhance adaptive immune response in myd 88 KO vs WT mice harboring B16 melanoma.Conclusion: The results of this study provide further evidence that blocking Myd 88 signaling increases anti tumor immunity against melanoma, and that the enhanced immunity can be explained, in part, by reduction of expression PDL1/PD1 immune checkpoint molecules. Considering the importance of tumor-infiltrating MDSCs in regulating anti tumor immunity in the tumor microenvironment, our findings could provide insight into the design of new therapeutics targeting Myd 88. Further experiments are needed to show how alteration in profile of PDL1 checkpoint expression on MDSCs influences anti-tumor T cell responses. DisclosuresNo relevant conflicts of interest to declare.

Innate signaling mechanisms controlling Mycobacterium chelonae-mediated CCL2 and CCL5 expression in macrophages

Mycobacterium chelonae (Mch) is an atypical rapidly growing mycobacterium (RGM) that belongs to the M. chelonae complex, which can cause a variety of human infections. During this type of mycobacterial infection, macrophage-derived chemokines play an important role in the mediation of intracellular communication and immune surveillance by which they orchestrate cellular immunity. However, the intracellular signaling pathways involved in the macrophage-induced chemokine production during Mch infections remain unknown. Thus, the present study aimed to determine the molecular mechanisms by which Mch activates the gene expressions of chemokine (C-C motif) ligand 2 (CCL2) and CCL5 in murine bone marrow-derived macrophages (BMDMs) and in vivo mouse model. Toll-like receptor 2 (TLR2)-deficient mice showed increased bacterial burden in spleen and lung and decreased protein expression of CCL2 and CCL5 in serum. Additionally, Mch infection triggered the mRNA and protein expression of CCL2 and CCL5 in BMDMs via TLR2 and myeloid differentiation primary response gene 88 (MyD88) signaling and that it rapidly activated nuclear factor (NF)-κB signaling, which is required for the Mch-induced expressions of CCL2 and CCL5 in BMDMs. Moreover, while the innate receptor Dectin-1 was only partly involved in the Mch-induced expression of the CCL2 and CCL5 chemokines in BMDMs, the generation of intracellular reactive oxygen species (ROS) was an important contributor to these processes. Taken together, the present data indicate that the TLR2, MyD88, and NF-κB pathways, Dectin-1 signaling, and intracellular ROS generation contribute to the Mch-mediated expression of chemokine genes in BMDMs.

Resveratrol Regulates Activated Hepatic Stellate Cells by Modulating NF-κB and the PI3K/Akt Signaling Pathway.

In the present study, we investigated whether resveratrol could suppress the hepatic fibrogenesis in activated hepatic stellate cells. The immortalized rat hepatic stellate cells, t-HSC/Cl-6, were treated with resveratrol 1 h prior to lipopolysaccharide (LPS, 1 μg/mL). Resveratrol decreased t-HSC/Cl-6 cell viability at much lower concentrations within 24 h. Resveratrol pretreatment also decreased the LPS-induced protein expression of α-SMA and collagen I. In addition, resveratrol significantly reduced the protein expression of Toll-like receptor 4 (TLR4) and myeloid differentiation primary response gene 88 (MyD88), and the expression of phosphorylated phosphatidylinositol 3-kinase (PI3K) and phosphorylated serine/threonine kinase B (Akt). Moreover, resveratrol markedly blocked the translocation of nuclear factor (NF)-κB in LPS-activated HSCs. Furthermore, resveratrol inhibited HSCs activation through stimulating LXRβ, but did not influence LXRα. Overall, we conclude that the antifibrotic effect of resveratrol is the result of blocking NF-κB activation and PI3K/Akt phosphorylation, which inhibits HSC activation to obstruct liver fibrosis. Thus, resveratrol may be a natural agent for preventing hepatic fibrosis.

Inhibitory effects of alternaramide on inflammatory mediator expression through TLR4-MyD88-mediated inhibition of NF-кB and MAPK pathway signaling in lipopolysaccharide-stimulated RAW264.7 and BV2 cells

Alternaramide (1), a novel lipophilic depsipeptide, has been isolated from the extract of the marine-derived fungus Alternaria sp. SF-5016. In the course of extensive biological evaluation of 1, its anti-inflammatory effects in lipopolysaccharide (LPS)-stimulated RAW264.7 and BV2 cells were observed. In our initial study of the anti-inflammatory effects of 1, the compound suppressed production of nitric oxide (NO) and prostaglandin E2 (PGE2) in LPS-stimulated RAW264.7 and BV2 cells. Suppression of NO and PGE2 production was correlated with the inhibitory effect of 1 on expression of LPS-induced inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) at the protein level in RAW264.7 and BV2 cells. In addition, 1 reduced production of pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6, and IL-12 in LPS-stimulated RAW264.7 and BV2 cells. In the evaluation of the molecular mechanisms underlying the anti-inflammatory effects of 1, the compound was found to suppress the nuclear factor-kappa B (NF-κB) signaling pathway in RAW264.7 and BV2 cells stimulated with LPS. This suppression was mediated by disruption of phosphorylation and degradation of IκBα, an inhibitor of NF-κB, in the cytoplasm, and blocking of nuclear translocation of the NF-κB p50–p65 heterodimer. Furthermore, 1 inhibited phosphorylation of c-Jun N-terminal kinases (JNKs) and p38 mitogen-activated protein kinase (MAPK), demonstrating its capacity to inhibit MAPK signaling. Finally, 1 markedly reduced expression of Toll-like receptor 4 (TLR4) and myeloid differentiation primary response gene 88 (MyD88) at the mRNA and protein levels in LPS-stimulated RAW264.7 and BV2 cells. Taken together, the results of the present study suggest that 1 modulates several TLR4-mediated inflammatory pathways, demonstrating its potential in the treatment of inflammatory and neuroinflammatory conditions.

Salvia miltiorrhiza extract protects white matter and the hippocampus from damage induced by chronic cerebral hypoperfusion in rats.

BackgroundSalvia miltiorrhiza (SM), an herbal plant, is traditionally used in the treatment of cardiovascular and cerebrovascular diseases in Asian countries. SM has multiple biological effects including anti-inflammatory activity. The present study is aimed at investigating the effects of SM extract in rats with chronic cerebral hypoperfusion.MethodsChronic cerebral hypoperfusion was induced in male Wistar rats by permanent bilateral common carotid artery occlusion (BCCAo). The rats were divided into 3 groups: sham-control, BCCAo treated with vehicle, and BCCAo treated with SM extract. Vehicle or SM extract (200 mg/kg) were administered daily by oral gavage beginning on day 21 after BCCAo and continuing to day 42. Immunohistochemical analyses were used to measure Iba-1-positive microglia and myelin basic protein (MBP) in white matter and hippocampal tissue. In addition, the expression levels of proinflammatory cytokines, including TNF-α, IL-1β, and IL-6, and the toll-like receptor (TLR) pathway in the hippocampus, were analyzed by western blot.ResultsAdministration of SM extract attenuated the activation of microglial cells in the white matter and hippocampus after BCCAo. SM extract also prevented neuroinflammation after BCCAo by reducing hippocampal levels of TNF-α, IL-1β, and IL-6, and increasing the reduced levels of MBP in the white matter and hippocampus. Further, the administration of SM extract alleviated the up-regulation of hippocampal TLR4 and myeloid differentiation primary response gene 88 (MyD88) in rats with chronic BCCAo.ConclusionsOur findings suggest that SM may be a promising therapeutic candidate in vascular dementia because of its protective effects against damage to the white matter and hippocampus after BCCAo.

Hypoxic stress: impact on the modulation of TLR2, TLR4, NOD1 and NOD2 receptor and their down-stream signalling genes expression in catla (Catla catla).

The damage-associated molecular patterns (DAMPs) released from the damaged tissue/cells are recently reported as endogenous ligands to activate toll-like receptors (TLRs) and nucleotide binding and oligomerization domain (NOD) receptors signaling pathways. In the aquatic environment, reduction in dissolved oxygen (DO) concentration causes hypoxic stress resulting in tissue damage and patho-biological changes in fish. We envisaged the critical role of TLR and NOD receptors in recognizing DAMPs as endogenous ligands during hypoxic stress in fish. Catla (Catla catla) fingerlings (avg. wt ~56g) was exposed to hypoxic stress (DO: 1-3mg/L) for 1 and 24h. After the designated time course, total RNA was extracted from gill, liver, kidney and blood, and modulation of TLRs (TLR2 and TLR4), NOD (NOD1 and NOD2) receptors, MyD88 (myeloid differentiation primary response gene 88), RICK (receptor interacting serine-threonine protein kinase-2), interleukin (IL)-6, IL-8 and IL-10 gene expression were analyzed by quantitative reverse transcriptase PCR assay. Significant (p<0.05) up-regulation of some DAMPs {high-mobility group box 1 and heat shock protein-70}, TLRs and NOD receptors genes expressions were observed in the hypoxic fish tissues as compared to the control. Further investigation revealed inductive expression of MyD88, RICK, IL-6, IL-8 and IL-10 genes in the TLRs and NODs activated tissues of the hypoxic fish. These data together may suggest the important role of TLRs and NOD receptors signaling pathway in sterile inflammation and pathobiology of fish in hypoxic stress, and warrant further study to investigate the role of TLR and NOD receptors in abiotic stress management in aquaculture.

Glatiramer acetate treatment negatively regulates type I interferon signaling.

Objective:Glatiramer acetate (GA; Copaxone), a disease-modifying therapy for multiple sclerosis (MS), promotes development of anti-inflammatory (M2, type II) monocytes that can direct differentiation of regulatory T cells. We investigated the innate immune signaling pathways that participate in GA-mediated M2 monocyte polarization.Methods:Monocytes were isolated from myeloid differentiation primary response gene 88 (MyD88)–deficient, Toll-IL-1 receptor domain–containing adaptor inducing interferon (IFN)–β (TRIF)–deficient, IFN-α/β receptor subunit 1 (IFNAR1)–deficient, and wild-type (WT) mice and human peripheral blood. GA-treated monocytes were stimulated with Toll-like receptor ligands, then evaluated for activation of kinases and transcription factors involved in innate immunity, and secretion of proinflammatory cytokines. GA-treated mice were evaluated for cytokine secretion and susceptibility to experimental autoimmune encephalomyelitis.Results:GA-mediated inhibition of proinflammatory cytokine production by monocytes occurred independently of MyD88 and nuclear factor–κB, but was blocked by TRIF deficiency. Furthermore, GA did not provide clinical benefit in TRIF-deficient mice. GA inhibited activation of p38 mitogen-activated protein kinase, an upstream regulator of activating transcription factor (ATF)–2, and c-Jun N-terminal kinase 1, which regulates IFN regulatory factor 3 (IRF3). Consequently, nuclear translocation of ATF-2 and IRF3, components of the IFN-β enhanceosome, was impaired. Consistent with these observations, GA inhibited production of IFN-β in vivo in WT mice, but did not modulate proinflammatory cytokine production by monocytes from IFNAR1-deficient mice.Conclusion:Our results demonstrate that GA inhibits the type I IFN pathway in M2 polarization of monocytes independently of MyD88, providing an important mechanism connecting innate and adaptive immune modulation in GA therapy and valuable insight regarding its potential use with other MS treatments.

Timosaponin B-II inhibits lipopolysaccharide-induced acute lung toxicity via TLR/NF-κB pathway

Timosaponin B-II (TB), a main bioactive compound in Anemarrhena asphodeloides Bunge, has various kinds of pharmacological activities, the present study aimed to investigate the protective role of TB on lipopolysaccharide (LPS)-induced acute lung injury (ALI). ALI was induced in mice by intratracheal instillation of LPS, and TB (20 and 60 mg/kg) was given orally 1 h prior to LPS administration. After 6 h, bronchoalveolar lavagefluid (BALF) and lung tissue were collected. TB decreased LPS-induced evident lung histopathological changes, lung wet-to-dry weight (W/D) ratio and lung myeloperoxidase (MPO) activity. In addition, TB inhibited inflammatory cells and cytokines including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) in BALF. Furthermore, we demonstrated that TB inhibited the Toll-like receptor-2 (TLR2), Toll-like receptor-4 (TLR4), myeloid differentiation primary response gene-88 (MyD88), nuclear factor-κB (NF-κB) p65 in LPS-induced ALI. These results showed that administration of TB prior to LPS improves ALI, possibly mediating ALI through suppressing TLR/NF-κB pathway activation.

Mycoplasma bovis-derived lipid-associated membrane proteins activate IL-1β production through the NF-κB pathway via toll-like receptor 2 and MyD88

Mycoplasma bovis causes pneumonia, otitis media, and arthritis in young calves, resulting in economic losses to the cattle industry worldwide. M. bovis pathogenesis results in part from excessive immune responses. Lipid-associated membrane proteins (LAMPs) can potently induce host innate immunity. However, interactions between M. bovis-derived LAMPs and Toll-like receptors (TLRs), or signaling pathways eliciting active inflammation and NF-κB activation, are incompletely understood. Here, we found that IL-1β expression was induced in embryonic bovine lung (EBL) cells stimulated with M. bovis-derived LAMPs. Subcellular-localization analysis revealed nuclear p65 translocation following EBL cell stimulation with M. bovis-derived LAMPs. An NF-κB inhibitor reversed M. bovis-derived LAMP-induced IL-1β expression. TLR2 and myeloid differentiation primary response gene 88 (MyD88) overexpression increased LAMP-dependent IL-1β induction. TLR2-neutralizing antibodies reduced IL-1β expression during LAMP stimulation. LAMPs also inhibited IL-1β expression following overexpression of a dominant-negative MyD88 protein. These results suggested that M. bovis-derived LAMPs activate IL-1β production through the NF-κB pathway via TLR2 and MyD88.

Effects of repeated handling and air exposure on the immune response and the disease resistance of gibel carp (Carassius auratus gibelio) over winter

High mortalities and suppressed immune functions of farmed fish over winter are the universal problems in aquaculture. It is necessary to improve the immune response and disease resistance in the overwintering fish. A recent study suggested that repeated handling increased innate immune mechanisms and disease resistance in Senegalese sole. Therefore, the present study evaluated the hypothesis that appropriate repeated handling could compromise the immune depression and increase the disease resistance in gibel carp over winter. The experiment was executed in field net cages (2 m × 2 m × 2 m) from Dec. 4, 2012 to Apr. 2, 2013. Three cages with 50 fish per cage were randomly designed as the control group and did not receive any interfere over winter. The other three cages received repeated handling with an air exposure for 5 min every week during the experiment. Fish were not fed over winter. At the end of the trial, fish were challenged with Aeromonas hydrophila at a dose of 1.5 × 10(8) CFU ml(-1). The results showed that no significant difference of final body weight was found between groups. The spleen and kidney somatic index increased in the control fish after bacterial challenge and showed a rising trend but not a statistical change in repeated handled fish. Plasma cortisol levels significantly increased in the control fish at 6 h post bacterial challenge and then declined. However, repeated handled fish did not show any significant change in plasma cortisol levels after challenge. The reduced inducement of heat shock protein 70 (HSP70) expressions by repeated handling was found in gibel carp post bacterial challenge. After overwintering, the repeated handled fish exhibited increased catalase (CAT) and superoxide dismutase (SOD) activities. Enhanced plasma CAT activities and reduced plasma malondialdehyde (MDA) contents were found in repeated handled fish over time against invading bacteria. Up-regulation of myeloid differentiation primary response gene 88 (MyD88) and interleukin 11 (IL11) was observed in repeated handled fish over time after bacterial challenge. The overexpression of IL11 was significantly reduced by repeated handling against invading bacteria compared to the control group. The present results implied that a MyD88-dependent signaling pathway was involved in the innate immune responses of gibel carp by repeated handling over winter against invading bacteria.