MyD88-dependent Signaling Pathway Research Articles

LYRM03, an ubenimex derivative, attenuates LPS-induced acute lung injury in mice by suppressing the TLR4 signaling pathway.

Toll-like receptor 4 (TLR4)-mediated signaling plays a critical role in sepsis-induced acute lung injury (ALI). LYRM03 (3-amino-2-hydroxy-4-phenyl-valyl-isoleucine) is a novel derivative of ubenimex, a widely used antineoplastic medicine. We previously found that LYRM03 has anti-inflammatory effects in cecal ligation puncture mouse model. In this study we determined whether LYRM03 attenuated LPS-induced ALI in mice. LPS-induced ALI mouse model was established by challenging the mice with intratracheal injection of LPS (5 mg/kg), which was subsequently treated with LYRM03 (10 mg/kg, ip). LYRM03 administration significantly alleviated LPS-induced lung edema, inflammatory cell (neutrophils and macrophages) infiltration and myeloperoxidase (MPO) activity, decreased pro-inflammatory and chemotactic cytokine (TNF-α, IL-6, IL-1β, MIP-2) generation and reduced iNOS and COX-2 expression in the lung tissues. In cultured mouse alveolar macrophages in vitro, pretreatment with LYRM03 (100 μmol/L) suppressed LPS-induced macrophage activation by reducing Myd88 expression, increasing IκB stability and inhibiting p38 phosphorylation. These results suggest that LYRM03 effectively attenuates LPS-induced ALI by inhibiting the expression of pro-inflammatory mediators and Myd88-dependent TLR4 signaling pathways in alveolar macrophages. LYRM03 may serve as a potential treatment for sepsis-mediated lung injuries.

Capsular Polysaccharide is a Main Component of Mycoplasma ovipneumoniae in the Pathogen-Induced Toll-Like Receptor-Mediated Inflammatory Responses in Sheep Airway Epithelial Cells.

Mycoplasma ovipneumoniae (M. ovipneumoniae) is characterized as an etiological agent of primary atypical pneumonia that specifically infects sheep and goat. In an attempt to better understand the pathogen-host interaction between the invading M. ovipneumoniae and airway epithelial cells, we investigated the host inflammatory responses against capsular polysaccharide (designated as CPS) of M. ovipneumoniae using sheep bronchial epithelial cells cultured in an air-liquid interface (ALI) model. Results showed that CPS derived from M. ovipneumoniae could activate toll-like receptor- (TLR-) mediated inflammatory responses, along with an elevated expression of nuclear factor kappa B (NF-κB), activator protein-1 (AP-1), and interferon regulatory factor 3 (IRF3) as well as various inflammatory-associated mediators, representatively including proinflammatory cytokines, such as IL1β, TNFα, and IL8, and anti-inflammatory cytokines such as IL10 and TGFβ of TLR signaling cascade. Mechanistically, the CPS-induced inflammation was TLR initiated and was mediated by activations of both MyD88-dependent and MyD88-independent signaling pathways. Of importance, a blockage of CPS with specific antibody led a significant reduction of M. ovipneumoniae-induced inflammatory responses in sheep bronchial epithelial cells. These results suggested that CPS is a key virulent component of M. ovipneumoniae, which may play a crucial role in the inflammatory response induced by M. ovipneumoniae infections.

Protective Effect of Phillyrin on Lethal LPS-Induced Neutrophil Inflammation in Zebrafish

Background/Aims: Forsythia suspensa Vahl. (Oleaceae) fruits are widely used in traditional Chinese medicine to treat pneumonia, typhoid, dysentery, ulcers and oedema. Antibacterial and anti-inflammatory activities have been reported for phillyrin (PHN), the main ingredient in Forsythia suspensa Vahl fruits, in vitro. However, the underlying mechanisms in vivo remain poorly defined. In this study, we discovered that PHN exerted potent anti-inflammatory effects in lethal LPS-induced neutrophil inflammation by suppressing the MyD88-dependent signalling pathway in zebrafish. Methods: LPS-yolk microinjection was used to induce a lethal LPS-infected zebrafish model. The effect of PHN on the survival of zebrafish challenged with lethal LPS was evaluated using survival analysis. The effect of PHN on neutrophil inflammation grading in vivo was assessed by tracking neutrophils with a transgenic line. The effects of PHN on neutrophil production and migration were analysed by SB⁺ cell counts during consecutive hours after modelling. Additionally, key cytokines and members of the MyD88 signalling pathway that are involved in inflammatory response were detected using quantitative RT-PCR. To assess gene expression changes during consecutive hours after modelling, the IL-1β, IL-6, TNF-α, MyD88, TRIF, ERK1/2, JNK, IκBa and NF-κB expression levels were measured. Results: PHN could protect zebrafish against a lethal LPS challenge in a dose-dependent manner, as indicated by decreased neutrophil infltration, reduced tissue necrosis and increased survival rates. Up-regulated IL-1β, IL-6 and TNF-α expression also showed the same tendencies of depression by PHN. Critically, PHN significantly inhibited the LPS-induced activation of MyD88, IκBa, and NF-κB but did not affect the expression of ERK1/2 MAPKs or JNK MAPKs in LPS-stimulated zebrafish. Additionally, PHN regulated the MyD88/IκBα/NF-κB signalling pathway by controlling IκBα, IL-1β, IL-6, and TNF-α expression. Conclusion: This study provides a rationale for the clinical application of PHN as an anti-inflammatory agent.

Molecular cloning and characterization of LrTLR4, analysis of its inductive expression and associated down-stream signaling molecules following lipopolysaccharide stimulation and Gram-negative bacterial infection

Toll-like receptors (TLRs) play key roles in innate immunity from lower to higher vertebrates. Among various TLR types, TLR4 was reported to recognize LPS in higher vertebrates resulting in the activation of down-stream signaling pathway. Except in some teleosts, function of TLR4 in most fish species including rohu (Labeo rohita) a commercially important fish species in the South-East Asian countries remained unknown. To investigate it, full-length cDNA of Labeo rohita TLR4 (LrTLR4) was cloned, and it consisted of 2729 bp, with a single ORF of 2469 bp encoding a polypeptide of 822 aa with a predicted molecular mass of 94.753 kDa. Structurally, LrTLR4 consisted of 25 LRRs (leucine rich repeat regions), one TM (trans-membrane) domain and one TIR (Toll/interleukin-1 receptor) domain, and was similar to higher vertebrate's TLR4. Phylogenetically, LrTLR4 exhibited highest (85%) identity with the common carp TLR4b amino acids sequence, and formed a separate subgroup in the phylogenetic tree. LrTLR4 was widely expressed in all tested organs/tissues, and amidst the tissues highest expression was detected in blood and the lowest in eye. In response to LPS-stimulation, LrTLR4 was induced with the activation of MyD88-dependent and TRIF-dependent signaling pathway resulting in pro-inflammatory cytokines (interleukin 6 and 8) and type I IFN gene expression. Infection of rohu with a Gram-negative fish pathogen (Aeromonas hydrophila), also activated LrTLR4. Together, these findings suggest the important role of TLR4 in LPS sensing and augmentation of innate immunity against Gram-negative bacterial infection in fish.

Lipopolysaccharide and IL-1β coordinate a synergy on cytokine production by upregulating MyD88 expression in human gingival fibroblasts.

Both lipopolysaccharide (LPS) and interleukin (IL)-1β activate the MyD88-dependent signaling pathways to stimulate proinflammatory cytokine expression. However, it remains unknown how LPS and IL-1β interact with each other to coordinate the stimulation. In this study, we sought to investigate the interaction between LPS and IL-1β on MyD88-dependent signaling pathways in human gingival fibroblasts (HGFs). Results showed that LPS derived from Porphyromonas gingivalis (Pg LPS) and IL-1β cooperatively stimulated mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NFκB) signaling pathways, and subsequent expression of proinflammatory cytokine expression. Furthermore, our results showed that Pg LPS and IL-1β exerted a synergy on MyD88 expression and knockdown of MyD88 expression by small interfering RNA diminished the synergistic effect of Pg LPS and IL-1β on IL-6 expression, suggesting that upregulation of MyD88 is involved in the coordinated stimulation by Pg LPS and IL-1β of proinflammatory cytokine expression. Finally, our results showed that pharmacological inhibitors for MAPK and NFκB significantly reduced IL-6 secretion stimulated by Pg LPS and IL-1β, indicating that the MyD88-dependent MAPK and NFκB signaling pathways are essential for the upregulation of proinflammatory cytokine expression by Pg LPS and IL-1β. Taken together, this study showed that LPS and IL-1β coordinate a synergy on cytokine production by upregulating MyD88 expression in HGFs.

Identification and expression analysis of IκB and NF-κB genes from Cyclina sinensis.

With the increasing economic importance of Cyclina sinensis aquaculture, interest in its defense mechanisms against pathogenic infection has grown in recent years. Inhibitor of nuclear factor-kappaB (IκB) and nuclear factor-kappaB (NF-κB) are proteins with central roles in many important physiological and pathological processes, such as innate immune responses. In this study, we identified CsIκB and CsNF-κB genes from a C.sinensis transcriptome library. In healthy adult clams, CsIκB and CsNF-κB genes were widely expressed in various tissues and highly expressed in hemocytes. Further, the expression levels of these genes were significantly increased in hemocytes challenged by Vibrio anguillarum, Micrococcus luteus and poly I:C. Inhibition of CsMyD88 expression by RNAi technology significantly altered the mRNA expression patterns of CsIκB and CsNF-κB as measured using quantitative real-time PCR. These results collectively indicated that the NF-κB signaling pathway, including CsIκB and CsNF-κB genes, might be involved in early innate immune responses and may be regulated by a MyD88-dependent signaling pathway in C.sinensis.

Zhikang Capsule ameliorates dextran sodium sulfate-induced colitis by inhibition of inflammation, apoptosis, oxidative stress and MyD88-dependent TLR4 signaling pathway

Ethnopharmacological relevanceZhikang Capsule (ZKC) is a traditional Chinese medicine (TCM) modified from classic formulas Qi-Li-San (an ancient formula dating to Qing Dynasty) and Fu-Jin-Sheng-Ji-San (written into The Golden Mirror of Medicine). ZKC contains 14 kinds of materials and has been widely used for the clinical therapy of inflammatory bowel diseases (IBD) for a long time. However, the therapeutic mechanisms of ZKC are still unclear. Aim of the studyTo determine the protective effect of ZKC on dextran sodium sulfate (DSS)-induced colitis and explore the underlying mechanisms. Materials and methodsC57BL/6 mice were fed with 3% DSS in drinking water for one week to induce experimental colitis. They were randomly assigned to six groups according to the treatment conditions. The histological changes of colon tissues were observed by hematoxylin and eosin (H&E) staining. The serum concentration of pro-inflammatory cytokines (TNF-α, IFN-γ, IL-1β, and IL-12) and anti-inflammatory mediators (IL-4 and IL-10) was detected by enzyme-linked immune sorbent assays (ELISAs). The production of MPO, SOD, MDA, NO, and caspase-3 was assessed by biochemical assay kits. The expression of iNOS, ICAM-1, and NF-ΚB was evaluated by immunohistochemistry staining. The levels of TLR4, MyD88, and TRAF6 were determined by western blot. ResultsHistologic analysis exhibited that ZKC alleviated the inflammation, loss of goblet cells, and submucosal edema induced by DSS. ZKC significantly suppressed the pro-inflammatory cytokines and promoted the anti-inflammatory mediators. The antioxidation of ZKC was indicated by increased activity of SOD and reduced production of MDA, NO, and iNOS in ZKC-treated mice. Furthermore, ZKC repressed the colonic expression of caspase-3 and the activity of the MyD88-dependent TLR4 signaling pathway. ConclusionsThis research demonstrated the protective effect of ZKC on DSS-induced colitis. For the first time, we identified four therapeutic mechanisms of ZKC, including effective inhibition of the inflammatory responses, significant alleviation of intestinal epithelium apoptosis, considerable prevention of oxidative stress, and selective down-regulation of the MyD88-dependent TLR4 signaling pathway. With high therapeutic effects and low toxic effects, ZKC exhibits great superiority over western medicines in IBD treatment.

Abstract 346: Potential role for R191, potent and selective IRAK4 kinase inhibitor, in treatment of hematologic malignancies

Abstract Recent advances in genome sequencing and tumor proteome and transcriptome analysis uncovered a key role for MyD88-dependent Toll-Like Receptor (TLR) and Interleukin-1 Receptor (IL-1R)-mediated signaling pathways in multiple hematologic malignancies. A third of activated B-cell-like diffuse large B-cell lymphoma (ABC DLBCL) and nearly 100% of Waldenstrom macroglobulinemia (WD) patients carry an activating Myd88 L265P mutation. Overexpression of multiple TLR pathway components, associated with deregulation of innate immune system and subsequent induction of proinflammatory bone marrow environment, are prominent features of myelodysplastic syndrome (MDS) and acute myelogenous leukemia (AML). IRAK4 kinase is a crucial enzyme in all MyD88-dependent signaling pathways, potentially making it an ideal target for disease modification with small molecule inhibitors. Through cell-based screening, we identified a potent small molecule IRAK1/4 kinase inhibitor, R191. R191 blocks TLR- and IL-1R-induced cytokine production in primary cells with potencies below 50nM while sparing unrelated pathways with at least 20-fold window. R191 is extremely potent in vitro against IRAK4 kinase (3 nM), yet exhibits good selectivity against a broad panel of kinases. In vivo, it decreases serum IL-6 in an acute mouse model of IL-1β-induced cytokine release and blocks joint inflammation in the collagen-induce arthritis model. R191 exhibited strong synergy with Bcl2 and BCR pathway inhibitors against a number of DLBCL lines in vitro. In multiple AML lines, R191 potently inhibited expression of PD-L1, potentially promoting recognition of AML blasts by the immune system. Based on the potential critical role of IRAK kinases in MDS, AML and Myd88 L265P lymphomas, R191 could provide a novel therapeutic modality in the management of multiple inflammation-driven hematologic malignancies. Citation Format: Vadim V. Markovtsov, Chrystelle Lamagna, Meagan Chan, Sothy Yi, Chi Young, Roy Frances, Stacey Siu, Sylvia Braselmann, Hui Li, Rajinder Singh, Gary Park, Esteban Masuda, Vanessa Taylor, Donald G. Payan. Potential role for R191, potent and selective IRAK4 kinase inhibitor, in treatment of hematologic malignancies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 346.

Desert dust induces TLR signaling to trigger Th2-dominant lung allergic inflammation via a MyD88-dependent signaling pathway

Asian sand dust (ASD) is known to exacerbate asthma, although its mechanism is not yet well understood. In this study, when the effects on inflammatory response by LPS present in ASD was investigated by measuring the gene expression of cytokines and chemokines in RAW264.7 cells treated with ASD and/or polymyxin B (PMB), the ASD effects were attenuated by PMB, but not completely. When an in vitro study was performed using bone marrow-derived macrophages (BMDMs) from WT, TLR2−/−, TLR4−/−, and MyD88−/− BALB/c mice and BMDMs from WT, TLR2−/−, TLR4−/−, TLR2/4−/−, TLR7/9−/−, and MyD88−/− C57BL/6J mice, cytokine (IL-6, IL-12) production in BMDMs was higher in ASD-stimulated TLR2−/− cells than in TLR4−/− cells, whereas it was lower or undetectable in TLR2/4−/− and MyD88−/− cells. These results suggest that ASD causes cytokine production predominantly in a TLR4/MyD88-dependent pathway. When WT and TLRs 2−/−, 4−/−, and MyD88−/− BALB/c mice were intratracheally challenged with OVA and/or ASD, ASD caused exacerbation of lung eosinophilia along with Th2 cytokine and eosinophil-relevant chemokine production. Serum OVA-specific IgE and IgG1 similar to WT was observed in TLRs 2−/−, 4−/− mice, but not in MyD88−/− mice. The Th2 responses in TLR2−/− mice were attenuated remarkably by PMB. These results indicate that ASD exacerbates lung eosinophilia in a MyD88-dependent pathway. TLRs 2 and 4 signaling may be important in the increase in lung eosinophilia. Also, the TLR4 ligand LPS and TLR2 ligand like β-glucan may be strong candidates for exacerbation of lung eosinophilia.

Molecular Characterization of MyD88 in Pinctada fucata and its Response to Lipopolysaccharides and Polyinosinic-cytidylic Acid

Myeloid differentiation factor 88 (MyD88) is a key and essential adapter involved in the interleukin-1 receptor (IL-1R) and toll-like receptor (TLR)-mediated activation signaling pathway. To investigate molecular characterization of MyD88 and its gene expression profile in response to stimulation by lipopolysaccharide (LPS) and polyinosinic-cytidylic acid (poly (I: C)), we isolated the MyD88 cDNA sequence in Pinctada fucata and analyzed expression patterns using quantitative real-time PCR. Sequence analysis indicated that Pf-MyD88 cDNA is 1463bp in length and contains a1050bp open reading frame that encodes a 349 α peptide. Pf-MyD88 has the highest similarity with homologues of Crassostrea gigas and highly conserved death and toll/IL-1R domains. Furthermore, during LPS and poly (I:C)-stimulated experiments in the gill, peak expression levels of Pf-MyD88 were detected at 2h and 8h with a 1.58-fold and 3.58-fold increase, respectively. The results revealed the existence of a MyD88-dependent signaling pathway in P. fucata and contributed to understanding the potential role of Pf-MyD88 in the TLR/IL-1R-mediated signaling pathway.

Fibronectin fragment-induced expression of matrix metalloproteinases is mediated by MyD88-dependent TLR-2 signaling pathway in human chondrocytes.

IntroductionFibronectin fragments (FN-fs) are increased in the cartilage of patients with osteoarthritis (OA) and have a potent chondrolytic effect. However, little is known about the cellular receptors and signaling mechanisms that are mediated by FN-fs. We investigated whether the 29-kDa amino-terminal fibronectin fragment (29-kDa FN-f) regulates cartilage catabolism via the Toll-like receptor (TLR)-2 signaling pathway in human chondrocytes.MethodsSmall interfering RNA was used to knock down TLR-2 and myeloid differentiation factor 88 (MyD88). TLR-2 was overexpressed in chondrocytes transfected with a TLR-2 expression plasmid. The expression levels of matrix metalloproteinase (MMP)-1, MMP-3, and MMP-13 were analyzed using quantitative real-time reverse transcription polymerase chain reactions, immunoblotting, or enzyme-linked immunosorbent assay. The effect of TLR-2 on 29-kDa FN-f-mediated signaling pathways was investigated by immunoblotting.ResultsTLR-2, TLR-3, TLR-4, and TLR-5 mRNA were significantly overexpressed in OA cartilage compared with normal cartilage, whereas no significant difference of TLR-1 mRNA expression was found. 29-kDa FN-f significantly increased TLR-2 expression in human chondrocytes in a dose- and time-dependent manner. Knockdown of TLR-2 or MyD88, the latter a downstream adaptor of TLR-2, significantly inhibited 29-kDa FN-f-induced MMP production at the mRNA and protein levels. Conversely, TLR-2 overexpression led to enhanced MMP production by 29-kDa FN-f. In addition, TLR-2 knockdown apparently inhibited 29-kDa FN-f-mediated activation of phosphorylated nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha, and p38, but not of c-Jun N-terminal kinase or extracellular signal-regulated kinase. Exposure to synovial fluid (SF) from affected joints of patients with OA elevated MMP-1, MMP-3, and MMP-13 expression markedly in primary chondrocytes without reducing cell viability. However, TLR-2 knockdown in chondrocytes significantly suppressed SF-induced MMP induction.ConclusionsOur data demonstrate that the MyD88-dependent TLR-2 signaling pathway may be responsible for 29-kDa FN-f-mediated cartilage catabolic responses. Our results will enhance understanding of cartilage catabolic mechanisms driven by cartilage degradation products, including FN-f. The modulation of TLR-2 signaling activated by damage-associated molecular patterns, including 29-kDa FN-f, is a potential therapeutic strategy for the prevention of cartilage degradation in OA.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-015-0833-9) contains supplementary material, which is available to authorized users.

Polysaccharopeptide exerts immunoregulatory effects via MyD88-dependent signaling pathway

AimsThe present study is to investigate the immunomodulatory mechanism and related pathways of polysaccharopeptide (PSP) in mice bearing Ehrlich's ascites carcinoma (EAC). MethodsTwelve female wild-type C57 mice were randomly divided into three groups. Another twelve female myeloid differentiation factor 88 (MyD88)-deficient mice were randomly assigned to three groups. Cell survival and peritoneal macrophage phagocytosis were measured using WST8 assay. Nitric oxide concentration was determined by Griess reaction. ELISA was used to measure tumor necrosis factor-α and interferon-γ levels. Quantitative real-time PCR was employed to measure mRNA levels. Western blotting was performed to determine protein expression. ResultsPSP significantly inhibited the proliferation of EAC cells via macrophage activation. PSP-primed macrophages exhibited a higher tumoricidal activity than untreated macrophages. PSP markedly inhibited the growth of the tumor and increased macrophage phagocytosis, nitric oxide release and cytokine secretion. Expression of MyD88 was markedly increased in PSP-treated groups, while ST2825 inhibited MyD88 signaling and interfered with nitric oxide release and the secretion of tumor necrosis factor-α and interferon-γ. Moreover, mRNA and protein levels associated with MyD88-dependent signaling pathway in MyD88-deficient mice group were significantly down-regulated compared with wild-type mice group. ConclusionsPSP plays an immunoregulatory effect through MyD88-dependent signaling pathway.

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.

Acute psychological stress induces short-term variable immune response

In spite of advances in understanding the cross-talk between the peripheral immune system and the brain, the molecular mechanisms underlying the rapid adaptation of the immune system to an acute psychological stressor remain largely unknown. Conventional approaches to classify molecular factors mediating these responses have targeted relatively few biological measurements or explored cross-sectional study designs, and therefore have restricted characterization of stress–immune interactions. This exploratory study analyzed transcriptional profiles and flow cytometric data of peripheral blood leukocytes with physiological (endocrine, autonomic) measurements collected throughout the sequence of events leading up to, during, and after short-term exposure to physical danger in humans. Immediate immunomodulation to acute psychological stress was defined as a short-term selective up-regulation of natural killer (NK) cell-associated cytotoxic and IL-12 mediated signaling genes that correlated with increased cortisol, catecholamines and NK cells into the periphery. In parallel, we observed down-regulation of innate immune toll-like receptor genes and genes of the MyD88-dependent signaling pathway. Correcting gene expression for an influx of NK cells revealed a molecular signature specific to the adrenal cortex. Subsequently, focusing analyses on discrete groups of coordinately expressed genes (modules) throughout the time-series revealed immune stress responses in modules associated to immune/defense response, response to wounding, cytokine production, TCR signaling and NK cell cytotoxicity which differed between males and females. These results offer a spring-board for future research towards improved treatment of stress-related disease including the impact of stress on cardiovascular and autoimmune disorders, and identifies an immune mechanism by which vulnerabilities to these diseases may be gender-specific.

Silkrose: A novel acidic polysaccharide from the silkmoth that can stimulate the innate immune response

We have identified a novel acidic polysaccharide from silkmoth (Antheraea yamamai) pupae that activates the mammalian innate immune response. This bioactive polysaccharide was isolated using nitric oxide production in mouse RAW264 macrophages as an indicator of immunostimulatory activity. We named this polysaccharide “silkrose”. It has a molecular weight of 3.15×105 and comprises nine monosaccharides. The expression profiles indicated that silkrose induced the expression of proinflammatory cytokines and interferon β that exist downstream of MyD88-dependent and MyD88-indeptendent signaling pathways. Also, the inhibition of Toll-like receptor 4 (TLR4), which exists upstream of the signaling pathways, led to the suppression of NO production by silkrose. Furthermore, this polysaccharide promoted the activation of nuclear factor kappa B in RAW264 cells, indicating that it stimulates the induction of various cytokines in macrophages through the TLR4 signaling pathway. Our results thus suggest that silkrose activates the innate immune response to various pathogenic microorganisms and viral infections.

Discovery of small molecule inhibitors of MyD88-dependent signaling pathways using a computational screen.

In this study, we used high-throughput computational screening to discover drug-like inhibitors of the host MyD88 protein-protein signaling interaction implicated in the potentially lethal immune response associated with Staphylococcal enterotoxins. We built a protein-protein dimeric docking model of the Toll-interleukin receptor (TIR)-domain of MyD88 and identified a binding site for docking small molecules. Computational screening of 5 million drug-like compounds led to testing of 30 small molecules; one of these molecules inhibits the TIR-TIR domain interaction and attenuates pro-inflammatory cytokine production in human primary cell cultures. Compounds chemically similar to this hit from the PubChem database were observed to be more potent with improved drug-like properties. Most of these 2nd generation compounds inhibit Staphylococcal enterotoxin B (SEB)-induced TNF-α, IFN-γ, IL-6, and IL-1β production at 2–10 μM in human primary cells. Biochemical analysis and a cell-based reporter assay revealed that the most promising compound, T6167923, disrupts MyD88 homodimeric formation, which is critical for its signaling function. Furthermore, we observed that administration of a single dose of T6167923 completely protects mice from lethal SEB-induced toxic shock. In summary, our in silico approach has identified anti-inflammatory inhibitors against in vitro and in vivo toxin exposure with promise to treat other MyD88-related pro-inflammatory diseases.

Dual Activation of TRIF and MyD88 Adaptor Proteins by Angiotensin II Evokes Opposing Effects on Pressure, Cardiac Hypertrophy, and Inflammatory Gene Expression.

Hypertension is recognized as an immune disorder whereby immune cells play a defining role in the genesis and progression of the disease. The innate immune system and its component toll-like receptors are key determinants of the immunologic outcome through their proinflammatory response. Toll-like receptor-activated signaling pathways use several adaptor proteins of which adaptor proteins myeloid differentiation protein 88 (MyD88) and toll-interleukin receptor domain-containing adaptor protein-inducing interferon-β (TRIF) define 2 major inflammatory pathways. In this study, we compared the contributions of MyD88 and TRIF adaptor proteins to angiotensin II (Ang II)-induced hypertension and cardiac hypertrophy in mice. Deletion of MyD88 did not prevent cardiac hypertrophy and the pressor response to Ang II tended to increase. Moreover, the increase in inflammatory gene expression (Tnfa, Nox4, and Agtr1a) was significantly greater in the heart and kidney of MyD88-deficient mice when compared with wild-type mice. Thus, pathways involving MyD88 may actually restrain the inflammatory responses. However, in mice with nonfunctional TRIF (Trif(mut) mice), Ang II-induced hypertension and cardiac hypertrophy were abrogated, and proinflammatory gene expression in heart and kidneys was unchanged or decreased. Our results indicate that Ang II induces activation of a proinflammatory innate immune response, causing hypertension and cardiac hypertrophy. These effects require functional adaptor protein TRIF-mediated pathways. However, the common MyD88-dependent signaling pathway, which is also activated simultaneously by Ang II, paradoxically exerts a negative regulatory influence on these responses.

Therapeutic potential of curcumin and curcumin analogues in rheumatology

Therapeutic potential of curcumin and curcumin analogues in rheumatology

Two myeloid differentiation factor 88 (MyD88) isoforms identified in ducks

MyD88 is an adaptor protein involved in the interleukin-1 receptor-induced and Toll-like receptor (TLR)-induced activation of nuclear factor-κB (NF-κB). In this study, we identified two isoforms of MyD88 gene, designated DuMyD88-X1 and DuMyD88-X2, from duck cells. Both variants were determined to have a death domain at the N-terminal and a Toll/IL-1R (TIR) domain at the C-terminal; however, the TIR domain of DuMyD88-X2 was incomplete and was 81 amino acids shorter than DuMyD88-X1. Quantitative real-time reverse transcription PCR revealed broad expression of both MyD88s. During Newcastle disease virus (NDV) challenge experiments, expression of the two genes increased significantly, with DuMyD88-X1 having a larger amplitude and longer duration. Overexpression of DuMyD88-X1 and DuMyD88-X2 induced the activation of NF-κB and IL-6 in vitro, suggesting that DuMyD88-X1 and DuMyD88-X2 may be important in the innate immune response. The results verify the existence of a MyD88-dependent signaling pathway in ducks and contribute to understanding the potential role of MyD88s in the innate immune response.

Interleukin-33 is a mediator of murine familial hemophagocytic lymphohistiocytosis (IRC5P.629)

Abstract Familial hemophagocytic lymphohistiocytosis (FHL) is a life-threatening disorder caused by uncontrolled, systemic immune activation. The murine model, in which lymphocytic choriomeningitis virus (LCMV) infection of perforin knockout (PKO) mice triggers FHL, demonstrates that the disease is driven by an excess of interferon-γ (IFNγ)-producing LCMV-specific CD8+ T cells. While this overactive T cell response is thought to arise from excess antigen stimulation through the TCR, data from our lab suggest that non-TCR, MyD88-dependent signaling pathways may be equally important. We show that interleukin-33 (IL-33), a cytokine released by damaged tissue, plays a critical role in driving FHL. LCMV-infected PKO mice receiving IL-33 receptor-blocking antibody (α-IL-33R) show markedly improved survival compared to isotype-treated controls (p=0.0005) and reduced severity of multiple disease parameters, including serum IFNγ (p=0.0005). α-IL-33R-treated mice have fewer gp33-specific CD8+ T cells (p=0.0044) and total effector CD8+ T cells (p<0.0001). IL-33R blockade also reduces the frequency and IFNγ MFI of IFNγ+ gp33-specific CD8+ T cells (p=0.0004 and p=0.0016, respectively). We observe similar effects on LCMV-specific CD4+ T cells, as well. These data demonstrate that disruption of IL-33 signaling in the murine model of FHL reduces T cell-mediated production of IFNγ, leading to improved morbidity and mortality, and suggest blockade of this pathway as a viable treatment strategy for FHL.