MyD88-dependent Signaling Pathway Research Articles

Network Pharmacology Analysis of the Mechanisms of Compound Herba Sarcandrae (Fufang Zhongjiefeng) Aerosol in Chronic Pharyngitis Treatment.

PurposeThis study aimed to investigate the molecular mechanisms of compound herba Sarcandrae aerosol, also known as the Fufang Zhongjiefeng (FFZJF) aerosol, in treating chronic pharyngitis (CP) using network pharmacology and in vivo experimental approaches.MethodsActive compounds and putative targets of five herbs in FFZJF were identified from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform, Chemistry Database, and Swiss Target Prediction databases. The therapeutic targets of CP were obtained from OMIM, Durgbank, DisGeNT, and GAD databases. The active compounds-target networks were constructed using Cytoscape 3.6.1. The overlapping targets of FFZJF active compounds and CP targets were further analyzed using the String database to construct protein–protein interaction (PPI) network. KEGG pathway and Gene Ontology enrichment analysis was performed using the Database for Annotation, Visualization, and Integrated Discovery. The predicted targets and pathways were validated in a group A β-hemolytic streptococcus-induced rat CP model.ResultsThere were 45 active compounds identified from FFZJF and 11 potential protein targets identified for CP treatment. PPI network demonstrated that IL6, PTGS2, TLR-4, and TNF may serve as the key targets of FFZJF for the treatment of CP. The main functional pathways involving these key targets include cytokine secretion, inflammatory response, MyD88-dependent toll-like receptor signaling pathway, toll-like receptor signaling pathway, TNF signaling pathway, and NF-κB signaling pathway. In a rat CP model, the elevation of serum TNF-α, IL1β, and IL6 levels, as well as the upregulation of TLR-4, MyD88, NF-κB P65 in the pharyngeal mucosal tissues could be effectively reduced by FFZJF treatment in a dose-dependent manner.ConclusionThrough a network pharmacology approach and animal study, we predicted and validated the active compounds of FFZJF and their potential targets for CP treatment. The results suggest that FFZJF can markedly alleviate GAS-induced chronic pharyngitis by modulating the TLR-4/MyD88/NF-κB signaling pathways.

High‐Altitude Exposure and Sustained Hypoxia Increase Expression of Inflammatory Genes and Enhance the Peripheral Blood Inflammatory Response to LPS

Transcriptional responses to hypoxia and inflammatory stimuli are linked via hypoxia inducible factor (HIF) and NF-kB crosstalk. Tissues experience hypoxia during infection or inflammatory states due to increased metabolic demand of immune cells, or impaired oxygen delivery by occluded blood supply or fluid buildup. However, while the evolutionarily conserved inflammatory responses to oxygen limitation are necessary for modulating the physiological responses to hypoxic stress, during chronic and/or systemic hypoxia, the crosstalk between these two pathways can become maladaptive. For example, hypoxia-induced inflammation may contribute to the development of Acute and Chronic Mountain Sickness. We hypothesized that acute sustained hypoxia exposure induces a systemic inflammatory response and changes sensitivity to subsequent inflammatory stimuli. To test this hypothesis, we used a high-altitude exposure model (1-3 days). We compared the RNA profiles in whole blood collected in the morning during fasting at sea level, and after 1 night and 3 nights at high altitude (3800m elevation) in 14 healthy sojourners (4 women, 10 men). RNA sequencing was coupled with Nanostring direct digital detection of 255 inflammatory pathway genes. 127 genes were significantly upregulated, and 140 genes were significantly downregulated with a fold change threshold > 1 and adjusted p-value < 0.05 on the first day at high altitude. Of all significant genes, 13 were also detected as significantly upregulated via Nanostring: HMGB1, TGFBR1, HMGN1, DDIT3, TLR8, MAPK1, TLR1, IFIT1, IFI44, MAPK8, HMGB2, TLR5, IFIT2 (adj. p < 0.05 for all). Among the significant gene ontology terms for all differentially expressed genes with adj p < 0.05 were: toll-like receptor signaling pathway (p<0.0001), MyD88-dependent toll-like receptor signaling pathway (p=0.012), interleukin-1-mediated signaling pathway (p=0.003), and I-kappaB kinase/NF-kappaB signaling (p<<0.0001). To test if upregulation of toll-like receptor signaling pathways increased sensitivity of peripheral blood mononuclear cells to inflammatory stimuli, we cultured whole blood and isolated monocytes in the presence of normoxia (18% O2, 5% CO2 at sea level) or hypoxia (1% O2, 5% CO2 at sea level) and with or without a lipopolysaccharide (LPS) stimulus for 24 hours. Both LPS (p<<0.0001) and hypoxia (p=0.01) independently increased TNF-a expression in whole blood and there was a significant interaction between hypoxia and LPS (p=0.002) in which hypoxia augmented TNF-a expression in response to LPS. TNF-a expression was higher in monocyte cultures exposed to LPS (p<<0.0001) and showed a nonsignificant trend to be higher in the presence of hypoxia, but there was no interaction. In conclusion, our results indicate that acute sustained hypoxia exposure may exacerbate the response to subsequent inflammatory stimuli. This may be particularly important in the context of pulmonary diseases if hypoxemia exacerbates the inflammatory response to viral or bacterial infection.

IL-12p40 and Type 1 immunity are induced by Toxoplasma gondii in the absence of the TLR-MyD88 signaling cascade

Abstract Toxoplasma gondii is an orally acquired pathogen that induces strong, protective IFN-γ based immunity. The response in mice is driven in part by well-characterized MyD88-dependent signaling pathways. The objective of this study was to determine the induction of immune responses that do not involve this Toll-like receptor (TLR)/IL-1 family receptor adaptor molecule, with particular focus on events in the intestinal mucosa. Using eYFP-IL-12p40 reporter mice on an MyD88−/− background, we identified dendritic cells, macrophages, and neutrophils as cellular sources of MyD88-independent IL-12 after peroral T. gondii infection. While infection-induced IL-12 was lower in the absence of MyD88, the cytokine was nonetheless required to induce type I immunity. Type 1 and type 3 innate lymphoid cells (ILC), CD4+ T cells, and CD8+ T cells each contributed to the IFN-γ pool. Furthermore, bacterial flagellin and Toxoplasma specific CD4+T cell populations in the lamina propria expanded to similar frequencies observed in the wildtype small intestine. ILC3 were expanded in both naïve and infected MyD88−/− mice relative to their MyD88+/+ counterparts, suggesting a compensatory response triggered by loss of MyD88. This study identifies MyD88-independent intestinal immune pathways induced by T. gondii including myeloid cell derived IL-12 production, downstream type I immunity, and IFN-γ production by ILC1, ILC3, and T lymphocytes. Collectively, these data reveal an underlying network of immune responses that do not involve MyD88 but that contribute to pathways associated with resistance to T. gondii.

Immunomodulatory effect of intracellular polysaccharide from mycelia of Agaricus bitorquis (QuéL.) Sacc. Chaidam by TLR4-mediated MyD88 dependent signaling pathway

Agaricus bitorquis (QuéL.) Sacc. Chaidam is a valuable edible fungus in Qinghai-Tibet plateau and ABSP is a novel intracellular polysaccharide from its mycelia. GC and NMR analysis determined ABSP is galactoglucomannan-like polysaccharide that may have immunomodulatory effect. This study used RAW264.7 as model cell to determine immunomodulatory effect of ABSP. After ABSP treatment, viability and phagocytic ability promoted, and NO, ROS, TNF-α levels also raised which proved ABSP had immune regulation to RAW264.7. WB and qRT-PCR determined the key proteins and genes expression of TLR4, MyD88, TRAF-6 and NF-κB significantly increased while protein and gene expression of TRAM had no significant increase. Also, TNF-α level extremely decreased by adding inhibitors of TLR4 and MyD88 which confirmed ABSP could immunologically regulate RAW264.7 byTLR4-MyD88 dependent pathway. This study would provide theoretical basis for further study on ABSP and be helpful for development of beneficial functionally foods and exploitation of this resource.

Supramolecular co-assembly of self-adjuvanting nanofibrious peptide hydrogel enhances cancer vaccination by activating MyD88-dependent NF-κB signaling pathway without inflammation

Peptide vaccine targeting tumor-specific antigens is a promising cancer treatment regimen. However, peptide vaccines are commonly low-immunogenic, leading to suboptimal antitumor T-cell responses. Current peptide vaccination approaches are challenged by the variability of peptide physicochemical characters and vaccine formulations, flexibility, and the broad feasibility. Here, the supramolecular co-assembly of antigen epitope-conjugated peptides (ECPs) targeting CD8 or CD4 T-cell receptors was used to engineer a nanofibrious hydrogel vaccine platform. This approach provided precise and tunable loading of peptide antigens in nanofibers, which notably increased the antigen uptake, cross-presentation, and activation of dendritic cells (DCs). Immunization in mice indicated that the co-assembled peptide hydrogel did not induce local inflammation responses and elicited significantly promoted T-cell immunity by activating the MyD88-dependent NF-κB signaling pathway in DCs. Vaccination of mice using co-assembled peptide vaccine stimulated both enhanced CD8 and CD4 T cells against EG.7-OVA tumors without additional immunoadjuvants or delivery systems, and resulted in a more remarkable cancer immunotherapy efficacy, compared with free peptide vaccine or aluminum-adjuvanted peptide formulation. Altogether, peptide co-assembly demonstrated by three independent pairs of ECPs is a facile, customizable, and chemically defined approach for co-delivering peptide antigens in self-adjuvanting hydrogel vaccines that could induce stronger anticancer T-cell responses.

Antimicrobial peptide temporin-1CEa isolated from frog skin secretions inhibits the proinflammatory response in lipopolysaccharide-stimulated RAW264.7 murine macrophages through the MyD88-dependent signaling pathway

Temporin-1CEa, which is isolated from the skin secretions of the Chinese brown frog Rana chensinensis, exhibits broad-spectrum antimicrobial activity against gram-positive and gram-negative bacteria and antitumor activity. LK2(6) and LK2(6)A(L) are the analogs of temporin-1CEa obtained by replacing amino acids and displayed an improved anticancer activity. In the present study, the anti-inflammatory activity and mechanism of action of temporin-1CEa and its analogs LK2(6) and LK2(6)A(L) in lipopolysaccharide (LPS)-stimulated RAW264.7 murine macrophages were investigated. The results showed that temporin-1CEa and its analogs decreased the production of the cytokines tumor necrosis factor-α and interleukin-6 by inhibiting the protein expression of nuclear factor-κB and mitogen-activated protein kinase and the MyD88-dependent signaling pathway. Isothermal titration calorimetry studies revealed that temporin-1CEa, LK2(6) and LK2(6)A(L) exhibited binding affinities to LPS, an important inflammatory inducer, with Kd values of 0.1, 0.03 and 0.06 μM, respectively. Circular dichroism and zeta potential experiments showed that temporin-1CEa and its analogs interacted with LPS by electrostatic binding between the positively charged peptides and negatively charged LPS, resulting in the neutralization of LPS toxicity.

R848 Is Involved in the Antibacterial Immune Response of Golden Pompano (Trachinotus ovatus) Through TLR7/8-MyD88-NF-κB-Signaling Pathway

R848 is an imidazoquinoline compound that is a specific activator of toll-like receptor (TLR) 7/8 and is often used in immunological research in mammals and teleosts. However, the immune responses initiated by R848 through the TLR7/8 pathway in response to bacterial infection remain largely unexplored in teleosts. In the current study, we investigated the antibacterial response and the participating signaling pathway initiated by R848 in golden pompano (Trachinotus ovatus). We found that R848 could stimulate the proliferation of head kidney lymphocytes (HKLs) in a dose-dependent manner, enhance the survival rate of HKLs, and inhibit the replication of bacteria in vivo. However, these effects induced by R848 were significantly reduced when chloroquine (CQ) was used to blocked endosomal acidification. Additionally, an in vivo study showed that R848 strengthened the antibacterial immunity of fish through a TLR7/8 and Myd88-dependent signaling pathway. A cellular experiment showed that Pepinh-MYD (a Myd88 inhibitor) significantly reduced the R848-mediated proliferation and survival of HKLs. Luciferase activity analysis showed that R848 enhanced the nuclear factor kappa B (NF-κB) activity, whereas this activity was reduced when CQ and Pepinh-MYD were present. Additionally, when an NF-κB inhibitor was present, the R848-mediated pro-proliferative and pro-survival effects on HKLs were significantly diminished. An in vivo study showed that knockdown of TLR7, TLR8, and Myd88 expression in golden pompano via siRNA following injection of R848 resulted in increased bacterial dissemination and colonization in fish tissues compared to that of fish injection of R848 alone, suggesting that R848-induced antibacterial immunity was significantly reduced. In conclusion, these results indicate that R848 plays an essential role in the antibacterial immunity of golden pompano via the TLR7/8-Myd88-NF-κB- signaling pathway.

Anti-inflammatory and antioxidant effects of Chaetoglobosin Vb in LPS-induced RAW264.7 cells: Achieved via the MAPK and NF-κB signaling pathways

There are few reports on the biological activities of chaetoglobosin Vb (Cha Vb) (a cytochalasin alkaloid). In this study, we investigated the molecular mechanisms underlying the anti-inflammatory and antioxidant effects of Cha Vb in the RAW264.7 cells stimulated lipopolysaccharide (LPS). LPS stimulation-induced oxidative stress (i.e. increase production of reactive oxygen species (ROS) and decreased expression of antioxidant superoxide dismutase (SOD)) was suppressed after a Cha Vb treatment. Cha Vb could significantly inhibit the upregulated expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) gene and protein induced by LPS whilst attenuating the production of pro-inflammatory cytokines TNF-α, IL-6 and IL-1β. Such antioxidant and anti-inflammatory effects were achieved through the TLR4-mediated MyD88-dependent signaling pathways (via suppressing the phosphorylation of p38, ERK, JNK MAPK and translocation of the NF-κB p65 subunit into nucleus), and the TRIF-dependent signaling pathways (via reducing IFN-β release without inhibiting interferon-regulated factor 3 (IRF3) and IRF7). At 25–100 μM (a concentration range with no cytotoxicity), Cha Vb dose-dependently influenced SOD enzyme activity and phosphorylation of p38, ERK1/2 and JNK, and at 100 μM, likely exerted the greatest inhibition towards LPS-induced oxidative stress and inflammatory response via the MAPK and NF-κB signaling pathway.

Nicotine Modulates MyD88-Dependent Signaling Pathway in Macrophages during Mycobacterial Infection.

Recently, we reported that cigarette smoking, and especially nicotine, increases susceptibility to mycobacterial infection and exacerbates inflammation in patients with Crohn’s disease (CD). The macrophagic response to Mycobacterium avium subspecies paratuberculosis (MAP) in CD and Mycobacteria tuberculosis (MTB) continues to be under investigation. The role of toll-like-receptors (TLRs) and cytoplasmic adaptor protein (MyD88) in proinflammatory response during Mycobacterial infection has been suggested. However, the mechanism of how nicotine modulates macrophage response during infection in CD and exacerbates inflammatory response remain unclear. In this study, we elucidated the mechanistic role of nicotine in modulating MyD88-dependent/TLR pathway signaling in a macrophage system during mycobacterial infection. The data demonstrated that MAP infection in THP-1 derived macrophages was mediated through TLR2 and MyD88 leading to increase in IL-8 in expression and production. On the other hand, LPS-representing, Gram-negative bacteria mediated macrophage response through TLR4. Blocking TLR2 and TLR4 with antagonists voided the effect of MAP, and LPS, respectively in macrophages and reversed response with decrease in expression of iNOS, TNF-α and IL-8. Interestingly, nicotine in infected macrophages significantly (1) downregulated TLR2 and TLR4 expression, (2) activated MyD88, (3) increased M1/M2 ratio, and (4) increased expression and secretion of proinflammatory cytokines especially IL-8, as seen in CD smokers. We also discovered that blocking macrophages during MAP infection with MyD88 antagonist significantly decreased response which illustrates the key role for MyD88 during infection. Surprisingly, dual treatment of MAP-infected macrophages with MyD88 antagonist and nicotine absolutely impaired immune response and decreased MAP viability, which clearly validate the inflammatory role of nicotine in macrophages through TLR2/MyD88 pathway during infection. This is the first report to describe the mechanism by which nicotine modulates TLR2/MyDD88 and exacerbates inflammation in CD smokers associated with infection.

Sargassum horneri (Turner) C. Agardh containing polyphenols attenuates particulate matter-induced inflammatory response by blocking TLR-mediated MYD88-dependent MAPK signaling pathway in MLE-12 cells

Ethnopharmacological relevanceSargassum horneri (Turner) C. Agardh (S. horneri), an edible brown marine algae, is known to have immunomodulatory effects and has been used in oriental medicine to treat inflammatory diseases. It is well known that ambient particulate matter (PM) is closely related to increased respiratory diseases inducing lung inflammation. AimConsidering the use of Sargassum horneri in traditional medicine to treat inflammatory diseases, we hypothesized and investigated the use of Sargassum horneri containing polyphenols against PM-induced inflammatory responses. Materials and methodsIn this study, we evaluated the impact of PM (majority <2.5 μm in diameter) on deep bronchial penetration ability upon inhalation and a therapeutic approach to mitigate its harmful effects using an ethanol extract of Sargassum horneri, an edible brown algae, containing polyphenols on a type II alveolar epithelial cell line, MLE-12. ResultsPM triggered mRNA expression of toll-like receptors (TLRs) TLR2/4/7, and those TLRs were significantly attenuated by Sargassum horneri extract (SHE). SHE further attenuated the phosphorylation of mitogen-activated protein kinase (MAPK) p38, extracellular signal-regulated kinase 1/2 (Erk1/2), and c-Jun NH (2)-terminal kinase (JNK), which were also activated in PM-exposed cells. Altogether, SHE subdued the PM-induced mRNA expression of pro-inflammatory cytokines (interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-6) and lung epithelial cell derived-chemokines (IL-8, monocyte chemoattractant protein-1 (MCP-1), and chemokine (C–C motif) ligand 5 (CCL5)). SHE also suppressed the mRNA expression of PM-induced pro-allergic cytokines thymic stromal lymphopoietin (TSLP) and interleukin (IL)-33. Furthermore, we showed that SHE suppressed the MAPK-dependent signaling pathway by attenuating receptor-associated factor (TRAF) 6 activation of proteins MyD88 and TNF. ConclusionTaking all the data together, we suggest that the anti-inflammatory potential of SHE on PM-exposed MLE-12 cells is mediated by the inhibition of PM-triggered downstream signaling along the TLR2/4/7–MyD88–TRAF6 axis of MAPK signaling.

Standardized ethanol extract of Tinospora crispa upregulates pro-inflammatory mediators release in LPS-primed U937 human macrophages through stimulation of MAPK, NF-\u03baB and PI3K-Akt signaling networks

BackgroundImmunomodulatory effects of Tinospora crispa have been investigated due to its traditional use to treat several inflammatory disorders associated to the immune system. The present study reports the underlying mechanisms involved in the stimulation of 80% ethanol extract of T. crispa stems on pro-inflammatory mediators release in lipopolysaccharide (LPS)-primed U937 human macrophages via MyD88-dependent pathways.MethodsRelease of interleukin (IL)-1β and tumor necrosis factor (TNF)-α, and production of prostaglandin E2 (PGE2) were determined by using enzyme-linked immunosorbent assay (ELISA). Immunoblot technique was executed to determine the activation of MAPKs molecules, NF-κB, PI3K-Akt and cyclooxygenase-2 (COX-2) protein. Determination of pro-inflammatory cytokines and COX-2 relative gene expression levels was by performing the real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). A reversed-phase HPLC method was developed and validated to standardize the T. crispa extract and chemical profiling of its secondary metabolites was performed by LC-MS/MS.ResultsQualitative and quantitative analyses of chromatographic data indicated that syringin and magnoflorine were found as the major components of the extract. T. crispa-treatment prompted activation of NF-κB by enhancing IKKα/β and NF-κB (p65) phosphorylation, and degradation of IκBα. The extract upregulated COX-2 protein expression, release of pro-inflammatory mediators and MAPKs (ERK, p38 and JNK) phosphorylation as well as Akt dose-dependently. T. crispa extract also upregulated the upstream signaling adaptor molecules, toll-like receptor 4 (TLR4) and MyD88. T. crispa-treatment also upregulated the pro-inflammatory markers mRNA expression.ConclusionThe results suggested that T. crispa extract stimulated the MyD88-dependent signaling pathways by upregulating the various immune inflammatory related parameters.

Effects of Mycobacterium vaccae vaccine in a mouse model of tuberculosis: protective action and differentially expressed genes

BackgroundTuberculosis is a leading cause of death worldwide. BCG is an effective vaccine, but not widely used in many parts of the world due to a variety of issues. Mycobacterium vaccae (M. vaccae) is another vaccine used in human subjects to prevent tuberculosis. In the current study, we investigated the potential mechanisms of M. vaccae vaccination by determining differentially expressed genes in mice infected with M. tuberculosis before and after M. vaccae vaccination.MethodsThree days after exposure to M. tuberculosis H37Rv strain (5 × 105 CFU), adult BALB/c mice randomly received either M. vaccae vaccine (22.5 μg) or vehicle via intramuscular injection (n = 8). Booster immunization was conducted 14 and 28 days after the primary immunization. Differentially expressed genes were identified by microarray followed by standard bioinformatics analysis.ResultsM. vaccae vaccination provided protection against M. tuberculosis infection (most prominent in the lungs). We identified 2326 upregulated and 2221 downregulated genes in vaccinated mice. These changes could be mapped to a total of 123 signaling pathways (68 upregulated and 55 downregulated). Further analysis pinpointed to the MyD88-dependent TLR signaling pathway and PI3K-Akt signaling pathway as most likely to be functional.ConclusionsM. vaccae vaccine provided good protection in mice against M. tuberculosis infection, via a highly complex set of molecular changes. Our findings may provide clue to guide development of more effective vaccine against tuberculosis.

Talin1 controls dendritic cell activation by regulating TLR complex assembly and signaling.

Talin critically controls integrin-dependent cell migration, but its regulatory role in skin dendritic cells (DCs) during inflammatory responses has not been investigated. Here, we show that talin1 regulates not only integrin-dependent Langerhans cell (LC) migration, but also MyD88-dependent Toll-like receptor (TLR)-stimulated DC activation. Talin1-deficient LCs failed to exit the epidermis, resulting in reduced LC migration to skin-draining lymph nodes (sdLNs) and defective skin tolerance induction, while talin1-deficient dermal DCs unexpectedly accumulated in the dermis despite their actomyosin-dependent migratory capabilities. Furthermore, talin1-deficient DCs exhibited compromised chemotaxis, NFκB activation, and proinflammatory cytokine production. Mechanistically, talin1 was required for the formation of preassembled TLR complexes in DCs at steady state via direct interaction with MyD88 and PIP5K. Local production of PIP2 by PIP5K then recruited TIRAP to the preassembled complexes, which were required for TLR signalosome assembly during DC activation. Thus, talin1 regulates MyD88-dependent TLR signaling pathways in DCs through a novel mechanism with implications for antimicrobial and inflammatory immune responses.

Induction of Th2 response through TLR2-mediated MyD88-dependent pathway in human microfold cells stimulated with chitosan nanoparticles loaded with Brucella abortus Mdh

Drug delivery by the nasal or oral route is considered the preferred route of administration because it can induce systemic mucosal immunity. However, few studies have examined the immunogenicity and transport of antigen at the level of the microfold (M) cell, the epithelial cell that specializes in antigen sampling at mucosal surfaces. In our previous study, Brucella abortus malate dehydrogenase (Mdh) was loaded in chitosan nanoparticles (CNs), and it induced high production of proinflammatory cytokines in THP-1 cells and systemic IgA in BALB/C mice. In the present study, an in vitro M cell model was used in which Caco-2 cells and Raji B cells were co-cultured to investigate the impact of the uptake and immunogenicity of B. abortus Mdh on nanoparticle transport in human M cells. Our results showed that loaded CNs induced enhanced transport of Mdh in the M cell model. ELISAs showed significantly higher production of IL-1β and IL-6 in the CN-Mdh stimulation group than that seen in the Mdh stimulation group. The observed increase of gene expression of TLR2, MyD88, TRAF6, IRF4 and CD14 implied that MyD88-dependent TLR2 signaling was activated by stimulation with CNs-Mdh. These results suggest that Mdh and CNs may function synergistically to enhance Th2-related responses triggered by the MyD88-dependent TLR2 signaling pathway and could induce an inflammatory response in M cells as an M cell-targeted delivery system. This study will contribute to the development of not only effective antigens for intracellular bacteria, including B. abortus, but also vaccine delivery systems that target M cells.

Fibrinogen-like protein 2 aggravates nonalcoholic steatohepatitis via interaction with TLR4, eliciting inflammation in macrophages and inducing hepatic lipid metabolism disorder.

Rationale: The functions of fibrinogen-like protein 2 (fgl2) have been studied in many inflammatory and neoplastic diseases, but the role of fgl2 in nonalcoholic fatty liver disease has not yet been elucidated. In this study, we sought to investigate the role of fgl2 in the pathogenesis of nonalcoholic steatohepatitis (NASH).Methods: Hepatic fgl2 expression was tested in patients with nonalcoholic fatty liver (NAFL) or NASH and controls. Wild-type and fgl2-/- C57BL/6 mice were subjected to a methionine/choline-deficient (MCD) diet or a high-fat diet (HFD) to establish NASH models. Bone marrow-derived macrophages (BMDMs) stimulated with LPS or free fatty acids were used for the in vitro study.Results: In both humans and mice with NASH, macrophage accumulation was concomitant with significantly increased fgl2 expression in the liver. Fgl2 deficiency attenuated liver steatosis and inflammation in diet-induced murine models of NASH. In both liver tissues and BMDMs from NASH mice, fgl2 deficiency resulted in reduced levels of proinflammatory cytokines and reactive oxygen species (ROS) compared with levels in wild-type controls. Activation of NF-κB, p38-MAPK and NLRP3 inflammasomes was also suppressed upon fgl2 disruption. Moreover, lipogenic genes (Fasn and SREBP-2) were downregulated while lipolytic genes (PPAR and CPT1A) were upregulated in the livers of fgl2-/- NASH mice. Primary hepatocytes incubated with the medium collected from fgl2-/- BMDMs showed less fat deposition than those incubated with WT BMDMs. Furthermore, we discovered that fgl2 combined with TLR4 mediates the activation of the Myd88-dependent signaling pathway, which may contribute to inflammation and lipid metabolism disorders.Conclusions: These data suggest that fgl2 aggravates the progression of NASH through activation of NF-κB, p38-MAPK and NLRP3 inflammasomes in macrophages, which consequently induces overproduction of proinflammatory cytokines and lipid metabolism disorders. An interaction of fgl2 and TLR4 may in part contribute to the activation of inflammatory signaling pathways in macrophages.

Waterborne microcystin-LR exposure induced chronic inflammatory response via MyD88-dependent toll-like receptor signaling pathway in male zebrafish

Waterborne microcystin-LR (MC-LR) released by cyanobacterial blooms in eutrophic water bodies have caused serious risk to aquatic animal and human health. In the present study, we for the first time conducted a comprehensive in vivo investigation on chronic inflammatory responses and its molecular pathways of different environmental relevant levels of MC-LR (0, 0.4, 2 and 10 μg/L) in male zebrafish (Danio rerio). The results showed that chronic MC-LR exposure caused splenic inflammatory changes including the formation of melano-macrophage centers, remarkable elevation of serum tumor necrosis factor alpha (TNFα) and interleukin 1 beta (IL1β) levels as well as significant upregulated expression of MyD88-dependent toll-like receptor (TLR/MyD88) signaling pathway genes (tlr4a, myd88, erk2, p38a, il1β and tnfα). The immunohistochemical and western blot results further validated that higher MC-LR concentrations tended to enhance the MyD88 signal. Moreover, significant decreases of serum C3 levels along with splenic c3b expression in the 10 μg/L exposure group proved that chronic MC-LR exposure could ultimately decrease the innate immunity of fish. Our findings revealed that chronic exposure of MC-LR could cause chronic inflammation through TLR/MyD88 signaling pathway and subsequently induce immune disorders in male zebrafish, which also urge us to pay more attention on the potential immunotoxicity of long-term exposure to low concentration of MC-LR.

The protection of indolealkylamines from LPS-induced inflammation in zebrafish

Ethnopharmacological relevanceToad skin came from Bufo bufo gargarizans Cantor and Bufo melanostictus Schneider. As the traditional Chinese medicine, it had the effect of clearing away heat and detoxification. In traditional applications, toad skin was often used for the treatment of cancer and inflammation. Total indolealkylamines (IAAs) from this medicine were proved the main compounds exert anti-inflammatory activity in our previous research. Aim of the studyIn the present study, we aimed to investigate the potential mechanism of anti-inflammatory activity of IAAs on LPS induced zebrafish. Materials and methodsLPS induced zebrafish was applicated as an in vivo inflammation model to clarify the structure-activity relationship of 4 major IAAs (N-methyl serotonin, bufotenine, dehydrobufotenine and bufothionine) from toad skin. Quantitative RT-PCR was applied to detect key cytokines and members of the MyD88-dependent signaling pathway. In addition, the targeted lipidomics was conducted to find out the potential biomarkers in the inflammatory zebrafish. Network pharmacology was used to unveil the main enzymes closely related to the target lipids. ResultsOur results showed that the anti-inflammatory activity of free IAAs (N-methyl serotonin, bufotenine and dehydrobufotenine) was more potent than that of combined IAAs (bufothionine). RT-PCR demonstrated that 4 IAAs exerted antiendotoxin inflammatory effect via suppressing the TLR4/MyD88/NF-κB and TLR4/MyD88/MAPKs signaling pathway. A total of 33 possible inflammatory biomarkers, including 14 SM, 6 Cer, 11 PC and 2 GlcCer, triggered by LPS were screened out. The levels of most of candidates could be regulated toward a normal level by IAAs, especially in N-methyl serotonin and dehydrobufotenine groups. Enzymes especially LBP, PLA2, CERK, SMPD and SGMS were found closely associated with the regulation of most lipid markers. ConclusionsOverall, the mechanism underlying the anti-inflammatory activity of IAAs probably attributed to their capability to suppress NF-κB and MAPKs inflammatory pathway. Meanwhile, IAAs could also interfere the metabolism of SM, Cer and PC probably by regulating LBP, PLA2, CERK, SMPD and SGMS.

Multiple Myd88-dependent Toll-like receptor signaling pathways mediate inflammation in a mouse model of primary Sjögren’s syndrome

Abstract Primary Sjögren’s syndrome (pSS) is an autoimmune disease characterized by exocrine gland dysfunction and immune hyperactivity. The adaptor Myd88 is critical for immune function, as most TLRs utilize Myd88 for signal transduction. Previous work by our group found Myd88 is required for pSS disease. Our objective was to identify the specific Myd88-dependent TLR pathways that mediate salivary and systemic inflammation. We used the pSS mouse model NOD.B10Sn-H2b/J (NOD.B10), Myd88-deficient NOD.B10 mice (NOD.B10Myd88−/−), and C57BL/10 controls. For all experiments we used female mice that were at least 26 weeks old, the age that NOD.B10 females display clinical disease. We isolated spleens from NOD.B10 mice and C57BL/10 controls and performed RNA-sequencing. We then harvested salivary tissue or spleens from NOD.B10, NOD.B10Myd88−/−, and C57BL/10 mice. We performed qPCR and flow cytometry to assess expression of Myd88-dependent TLRs. We cultured salivary tissue from NOD.B10 and NOD.B10Myd88−/− mice and performed cytokine arrays on the supernatants. Finally, we treated salivary tissue with a TLR4 inhibitor and quantified spontaneous IL-6 secretion. We identified dysregulation of numerous TLR-related networks in pSS splenocytes. We found upregulation of Myd88 dependent-TLRs in both the spleen and salivary tissue from NOD.B10 mice. Salivary tissue from Myd88-sufficient NOD.B10 females exhibited spontaneous secretion of IL-6, MCP-1 and TNFa, and this was diminished in NOD.B10Myd88−/− mice. Finally, TLR4 blockade reduced IL-6 secretion in NOD.B10 salivary tissue. Our preliminary data suggest Myd88-dependent pathways contribute to the inflammatory landscape in pSS, and inhibition of such will likely have therapeutic utility.

Effects of restrictions on maternal feed intake on the immune indexes of umbilical cord blood and liver Toll-like receptor signaling pathways in fetal goats during pregnancy

BackgroundLiver has important immune function during fetal development and after birth. However, the effect of maternal malnutrition on immune function of the fetal liver is rarely reported. In this study, twelve pregnant goats (Xiangdong black goat, at d 45 of gestation) were assigned to the control group (fed 100% of nutritional requirements) and the restriction group (fed 60% of the intake of the control group) during gestation from d 55 to 100. Fetal goats were harvested at d 100 of gestation and immune indexes and amino acid profiles of the umbilical cord blood and liver Toll-like receptors (TLRs) signaling pathways were measured.ResultsMaternal body weight in the restriction group was lower than the control group (P < 0.05). Maternal feed intake restriction decreased (P < 0.05) heart weight, heart index, alkaline phosphatase and serum amyloid protein A in the umbilical cord blood (UCB). Moreover, only histidine was decreased in the restricted group (P = 0.084), and there were no differences in other amino acids contents in the UCB between the two groups (P > 0.05). The TLR2 and TLR4 mRNA expression in the fetal liver in the restriction group was greater (P < 0.05) than that in the control group. Furthermore, the mRNA expression levels of myeloid differentiation primary response 88 (MyD88), TNF receptor associated factor 6, nuclear factor kappa B subunit 1, NFKB inhibitor alpha, IFN-β, TGF-β, TNF-α and IL-1β in the restricted group were upregulated (P < 0.05), and the expression of TLR3 (P = 0.099) tended to be higher in the restricted group. However, protein levels of TLR2, TLR4, IκBα, phosphorylated IκBα, phosphorylated IκBα/total IκBα, TRIF and MyD88 were not affected (P > 0.05) by maternal intake restriction.ConclusionsThese results revealed that the restriction of maternal feed intake influenced the development of heart and hepatic protein synthesis at the acute phase of fetal goats and upregulated the mRNA expression of genes involved in MyD88-dependent signaling pathways and of target cytokines.

Interleukin-1 alpha increases anti-tumor efficacy of cetuximab in head and neck squamous cell carcinoma

BackgroundDespite the high prevalence of epidermal growth factor receptor (EGFR) overexpression in head and neck squamous cell carcinomas (HNSCCs), incorporation of the EGFR inhibitor cetuximab into the clinical management of HNSCC has not led to significant changes in long-term survival outcomes. Therefore, the identification of novel therapeutic approaches to enhance the clinical efficacy of cetuximab could lead to improved long-term survival for HNSCC patients. Our previous work suggests that EGFR inhibition activates the interleukin-1 (IL-1) pathway via tumor release of IL-1 alpha (IL-1α), although the clinical implications of activating this pathway are unclear in the context of cetuximab therapy. Given the role of IL-1 signaling in anti-tumor immune response, we hypothesized that increases in IL-1α levels would enhance tumor response to cetuximab.MethodsParental and stable myeloid differentiation primary response gene 88 (MyD88) and IL-1 receptor 1 (IL-1R1) knockdown HNSCC cell lines, an IL-1R antagonist (IL-1RA), neutralizing antibodies to IL-1α and IL-1β, and recombinant IL-1α and IL-1β were used to determine cytokine production (using ELISA) in response to cetuximab in vitro. IL-1 pathway modulation in mouse models was accomplished by administration of IL-1RA, stable overexpression of IL-1α in SQ20B cells, administration of rIL-1α, and administration of a polyanhydride nanoparticle formulation of IL-1α. CD4+ and CD8+ T cell-depleting antibodies were used to understand the contribution of T cell-dependent anti-tumor immune responses. Baseline serum levels of IL-1α were measured using ELISA from HNSCC patients treated with cetuximab-based therapy and analyzed for association with progression free survival (PFS).ResultsCetuximab induced pro-inflammatory cytokine secretion from HNSCC cells in vitro which was mediated by an IL-1α/IL-1R1/MyD88-dependent signaling pathway. IL-1 signaling blockade did not affect the anti-tumor efficacy of cetuximab, while increased IL-1α expression using polyanhydride nanoparticles in combination with cetuximab safely and effectively induced a T cell-dependent anti-tumor immune response. Detectable baseline serum levels of IL-1α were associated with a favorable PFS in cetuximab-based therapy-treated HNSCC patients compared to HNSCC patients with undetectable levels.ConclusionsAltogether, these results suggest that IL-1α in combination with cetuximab can induce a T cell-dependent anti-tumor immune response and may represent a novel immunotherapeutic strategy for EGFR-positive HNSCCs.