IL1A regulates the inflammation in gout through the Toll-like receptors pathway.

Objective: Gout is a dangerous metabolic condition related to monosodium urate (MSU). Our aim is to study the molecular mechanisms underlying gout and to identify potential clinical biomarkers by bioinformatics analysis and experimental validation. Methods: In this study, we retrieved the overlapping genes between GSE199950-Differential Expressed Genes (DEGs) dataset and key module in Weighted Gene Co-Expression Network Analysis (WGCNA) on GSE199950. These genes were then analyzed by protein-protein interaction (PPI) network, expression and Gene Set Enrichment Analysis to identify the hub gene related to gout. Then, the gene was investigated by peripheral blood mononuclear cells (PBMCs), immunoassay and cell experiments like western blotting to uncover its underlying mechanism in gout cells. Results: From the turquoise module and 83 DEGs, we identified 62 overlapping genes, only 11 genes had mutual interactions in PPI network and these genes were highly expressed in MSU-treated samples. Then, it was found that the IL1A (interleukin 1 alpha) was the only one gene related to Toll-like receptor signaling pathway that was associated with the occurrence of gout. Thus, IL1A was determined as the hub gene in this study. In immunoassay, IL1A was significantly positively correlated with B cells and negatively correlated with macrophages. Moreover, IL1A is highly expressed in gout patients,it has a good clinical diagnostic value. Finally, the results of in vitro experiments showed that after knocking down IL1A, the expressions of pro-inflammatory cytokines and Toll-like receptor signaling pathway-related proteins (TLR2, TLR4, MyD88) were all reduced. Conclusion: It is confirmed that IL1A is a promoting gene in gout with a good diagnostic value, and specifically it affects the inflammation in gout through Toll-like receptor pathway. Our research offers fresh perspectives on the pathophysiology of gout and valuable directions for future diagnosis and treatment.

Expression of Toll-Like Receptors in the Lung Tissue of Mouse Fetuses Generated by in vitro Embryo Culture and Embryo Transfer.

Mouse fetuses generated by in vitro embryo culture and embryo transfer exhibit impaired lung development, altered composition of pulmonary epithelial cells associated with downregulation of several genes involved in lung development and toll-like receptor (TLR) signaling pathway. The aims of the present study were to determine the expression of all TLRs and to examine if the expression of TLRs, along with genes involved in TLR signaling pathway, is altered in the lung tissue of mouse fetuses generated through embryo culture and embryo transfer. Two experimental (EGs) and one control (CG) group were included in the study. Embryos cultured at 5% CO2-95% air for 95 h or less than 24 h were transferred to pseudo-pregnant females to obtain fetuses comprising EGin vitro (n = 18) and EGin vivo (n = 18), respectively. Fetuses obtained from naturally ovulating females on day 18 of pregnancy served as the CG (n = 18). Western blot and immunohistochemistry were used to determine the expression of TLR proteins. The expression of transcripts encoding TLRs, and the genes involved in TLR signaling pathway (Lbp, Pik3r1, Pik3cb, Nfkbia, and Fos), was determined using qRT-PCR. While all TLRs were expressed by cells lining the bronchial/bronchiolar epithelium of lung tissues in all groups, some of the TLRs were expressed in a specific pattern. When compared to CG, the expression of transcripts encoding TLR-2, -3, -4, -5, -7, -8, -9, -12, -13, Lbp, Pik3r1, Pik3cb, Nfkbia, and Fos was significantly downregulated in both EGs. It appears that stress imposed on embryos at preimplantation stages of development is associated with downregulation of TLRs, along with some of the genes involved in TLR signaling pathway, in the lung tissue during the perinatal period. It remains to be determined if downregulation of TLRs, along with the genes involved in TLR signaling pathway, has any functional consequences in the adult lung tissue.

Toll-like receptors 2 and 4 stress signaling and sodium-glucose cotransporter-2 in kidney disease.

Kidney disease is the 6th fastest-growing cause of death and a serious global health concern that urges effective therapeutic options. The inflammatory response is an initial reaction from immune and parenchymal cells in kidney diseases. Toll-like receptors (TLR) 2 and 4 are highly expressed by various kidney cells and respond to 'signaling danger' proteins, such as high mobility group box binding protein 1 (HMGB1) and prompt the progression of kidney disease by releasing inflammatory mediators. Burgeoning reports suggest that both SGLT2 and ER stress elevates TLR2/4 signaling via different axis. Moreover, SGLT2 signaling aggravates inflammation under the disease condition by promoting the NLR family pyrin domain-containing three inflammasomes and ER stress. Intriguingly, TLR2/4 downstream adaptors activate ER stress regulators. The above-discussed interactions imply that TLR2/4 does more than immune response during kidney disease. Here, we discuss in detail evidence of the roles and regulation of TLR2/4 in the context of a relationship between ER stress and SGLT2. Also, we highlighted different preclinical studies of SGLT2 inhibitors against TLR2/4 signaling in various kidney diseases. Moreover, we discuss the observational and interventional evidence about the relation between TLR2/4, ER stress, and SGLT2, which may represent the TLR2/4 as a potential therapeutic target for kidney disease.

Imbalanced Inflammatory Responses in Preterm and Term Cord Blood Monocytes and Expansion of the CD14+CD16+ Subset upon Toll-like Receptor Stimulation.

Developmentally regulated features of innate immunity are thought to place preterm and term infants at risk of infection and inflammation-related morbidity. Underlying mechanisms are incompletely understood. Differences in monocyte function including toll-like receptor (TLR) expression and signaling have been discussed. Some studies point to generally impaired TLR signaling, others to differences in individual pathways. In the present study, we assessed mRNA and protein expression of pro- and anti-inflammatory cytokines in preterm and term cord blood (CB) monocytes compared with adult controls stimulated ex vivo with Pam3CSK4, zymosan, polyinosinic:polycytidylic acid, lipopolysaccharide, flagellin, and CpG oligonucleotide, which activate the TLR1/2, TLR2/6, TLR3, TLR4, TLR5, and TLR9 pathways, respectively. In parallel, frequencies of monocyte subsets, stimulus-driven TLR expression, and phosphorylation of TLR-associated signaling molecules were analyzed. Independent of stimulus, pro-inflammatory responses of term CB monocytes equaled adult controls. The same held true for preterm CB monocytes-except for lower IL-1ß levels. In contrast, CB monocytes released lower amounts of anti-inflammatory IL-10 and IL-1ra, resulting in higher ratios of pro-inflammatory to anti-inflammatory cytokines. Phosphorylation of p65, p38, and ERK1/2 correlated with adult controls. However, stimulated CB samples stood out with higher frequencies of intermediate monocytes (CD14+CD16+). Both pro-inflammatory net effect and expansion of the intermediate subset were most pronounced upon stimulation with Pam3CSK4 (TLR1/2), zymosan (TR2/6), and lipopolysaccharide (TLR4). Our data demonstrate robust pro-inflammatory and yet attenuated anti-inflammatory responses in preterm and term CB monocytes, along with imbalanced cytokine ratios. Intermediate monocytes, a subset ascribed pro-inflammatory features, might participate in this inflammatory state.

Immune Activation and Inflammatory Response Mediated by the NOD/Toll-like Receptor Signaling Pathway-The Potential Mechanism of Bullfrog (Lithobates catesbeiana) Meningitis Caused by Elizabethkingia miricola.

Elizabethkingia miricola is an emerging opportunistic pathogen that is highly pathogenic in both immunocompromised humans and animals. Once the disease occurs, treatment can be very difficult. Therefore, a deep understanding of the pathological mechanism of Elizabethkingia miricola is the key to the prevention and control of the disease. In this study, we isolated the pathogenic bacteria from bullfrogs with dark skin color, weak limbs, wryneck, and cataracts. Via subsequent morphological observations and a 16S rRNA gene sequence analysis, the pathogen was identified as Elizabethkingia miricola. The histopathological and transmission electron microscopy analysis revealed that the brain was the main target organ. Therefore, brain samples from diseased and healthy bullfrogs were used for the RNA-Seq analysis. The comparative transcriptome analysis revealed that the diseased bullfrog brain was characterized by the immune activation and inflammatory response, which were mediated by the "NOD-like receptor signaling pathway" and the "Toll-like receptor signaling pathway". We also performed qRT-PCR to examine the expression profile of inflammation-related genes, which further verified the reliability of our transcriptome data. Based on the above results, it was concluded that the NOD/Toll-like receptor-related networks that dominate the immune activation and inflammatory response were activated in the brain of Elizabethkingia miricola-infected bullfrogs. This study contributes to the search for therapeutic targets for bullfrog meningitis and provides basic information for establishing effective measures to prevent and control bullfrog meningitis.

Bacterial Quorum-Sensing Signal DSF Inhibits LPS-Induced Inflammations by Suppressing Toll-like Receptor Signaling and Preventing Lysosome-Mediated Apoptosis in Zebrafish.

Bacteria and their eukaryotic hosts have co-evolved for millions of years, and the former can intercept eukaryotic signaling systems for the successful colonization of the host. The diffusible signal factor (DSF) family represents a type of quorum-sensing signals found in diverse Gram-negative bacterial pathogens. Recent evidence shows that the DSF is involved in interkingdom communications between the bacterial pathogen and the host plant. In this study, we explored the anti-inflammatory effect of the DSF and its underlying molecular mechanism in a zebrafish model. We found that the DSF treatment exhibited a strong protective effect on the inflammatory response of zebrafish induced by lipopolysaccharide (LPS). In the LPS-induced inflammation zebrafish model, the DSF could significantly ameliorate the intestinal pathological injury, reduce abnormal migration and the aggregation of inflammatory cells, inhibit the excessive production of inflammatory mediator reactive oxygen species (ROS) content, and prevent apoptosis. Through an RNA-Seq analysis, a total of 938 differentially expressed genes (DEGs) was screened between LPS and LPS + DSF treatment zebrafish embryos. A further bioinformatics analysis and validation revealed that the DSF might inhibit the LPS-induced zebrafish inflammatory response by preventing the activation of signaling in the Toll-like receptor pathway, attenuating the expression of pro-inflammatory cytokines and chemokines, and regulating the activation of the caspase cascade through restoring the expression of lysosomal cathepsins and apoptosis signaling. This study, for the first time, demonstrates the anti-inflammatory role and a potential pharmaceutical application of the bacterial signal DSF. These findings also suggest that the interkingdom communication between DSF-producing bacteria and zebrafish might occur in nature.

[Exploration of mechanism of "simultaneous treatment of brain and heart" of Naoxintong Capsules based on Toll-like receptor signaling pathway].

This study aims to explore the mechanism of "simultaneous treatment of the brain and the heart" of Naoxintong Capsules(NXT) under cerebral ischemia based on Toll-like receptor(TLR) signaling pathway.Male SD rats were randomized into sham operation group, model group, NXT group, and positive drug group.Middle cerebral artery occlusion(MCAO) model rats were used in model group, NXT group, and positive drug group, respectively.Neurological function was scored with the Bederson scale, and brain infarct rate was measured by 2,3,5-triphenyltetrazolium chloride(TTC) staining.Brain edema was detected with wet-dry weight method.Hematoxylin-eosin(HE) staining and TdT-mediated dUTP nick-end labeling(TUNEL) staining were used to observe and count apoptotic cardiocytes.In addition, serum myocardial enzymes were measured.The expression of 8 TLR signaling pathway-related proteins interferon-α(IFN-α), interferon regulatory factor-3(IRF3), interferon regulatory factor-7(IRF7), TLR2, TLR4, TLR7, TLR9, and tumor necrosis factor-α(TNF-α) in the cerebral cortex and heart of rats was detected by Western blot. Brain infarct rate, neurological function score, and brain water content in NXT group decreased significantly compared with those in the model group. At the same time, the reduction in apoptosis rate of cardiocytes and the content of serum aspartate aminotransferase(AST), alanine aminotransferase(ALT), creatine kinase(CK), and lactate dehydrogenase(LDH) were decreased in the NXT group.Systems pharmacological results and previous research showed that TLR signaling pathway played an important role in immune inflammatory response.The study of TLR signaling pathway and related proteins is helpful to elucidate the mechanism of "simultaneous treatment of the brain and the heart". Western blot results showed that NXT significantly inhibited the expression of IRF3, IRF7, TLR2, TLR7, and TNF-α in cerebral cortex and heart under cerebral ischemia.Cerebral ischemia influences cardiac functions, and TLR signaling pathway is one of the pathways for "simultaneous treatment of the brain and the heart" of NXT.

Activation of toll-like receptor signaling in endothelial progenitor cells dictates angiogenic potential: from hypothesis to actual state.

Human endothelial progenitor cells (EPCs) were isolated from cord blood samples and enriched by magnetic activated cell sorting method based on the CD133 marker. Cells were incubated with different doses of bacterial lipopolysaccharide, ranging from 2, 5, 10, 50, 100, 200, 250, 500, to 1000 µg/ml, for 48 h. The cell survival rate was determined by using MTT assay. To confirm activation of the toll-like receptor signaling pathway, PCR array analysis was performed. Protein levels of ERK1/2, p-ERK1/2, NF-ƙB and TRIF proteins were measured using western blotting. The content of TNF-α and lipoprotein lipase activity were analyzed by immunofluorescence imaging. Flow cytometric analysis of CD31 was performed to assess the maturation rate. Cell migration was studied by the Transwell migration assay. The expression of genes related to exosome biogenesis was measured using real-time PCR analysis. In vivo gel plug angiogenesis assay was done in nude mice. Lipopolysaccharide changed endothelial progenitor cells' survival in a dose-dependent manner with maximum viable cells in groups treated with 2 µg/ml. PCR array analysis showed the activation of toll-like signaling pathways after exposure to LPS (p<0.05). Western blotting analysis indicated an induction of p-ERK1/2 and Erk1/2, NF-kB and TRIF in LPS-treated EPCs compared with the control (p<0.05). Immunofluorescence staining showed an elevation of TNF-α and lipoprotein lipase activity after lipopolysaccharide treatment (p<0.05). Lipopolysaccharide increased EPC migration and expression of exosome biogenesis-related genes (p<0.05). In vivo gel plug analysis revealed enhanced angiogenesis in cells exposed to bacterial lipopolysaccharide. Data highlighted the close relationship between the toll-like receptor signaling pathway and functional activity in EPCs.

Is there a relationship between psoriasis and hepatitis C? A meta-analysis and bioinformatics investigation.

BACKGROUND: The relationship between psoriasis and hepatitis C was previously controversial, so our purpose is to investigate this connection. METHODS: We conducted a systematic review of the case-control, cross-sectional and cohort studies examining the association between psoriasis and hepatitis C in PubMed, EMBASE and Cochrane library databases and investigated the overlapping genes between psoriasis targets and hepatitis C targets using bioinformatics analysis. Based on overlapping genes and hub nodes, we also constructed the protein-protein interaction (PPI) network and module respectively, followed by the pathway enrichment analysis. RESULTS: We included 11 publications that reported a total of 11 studies (8 cross-sectional and 3 case-control). The case-control and cross-sectional studies included 25,047 psoriasis patients and 4,091,631 controls in total. Psoriasis was associated with a significant increase of prevalent hepatitis C (OR 1.72; 95% confidence interval [CI] (1.17-2.52)). A total of 389 significant genes were common to both hepatitis C and psoriasis, which mainly involved IL6, TNF, IL10, ALB, STAT3 and CXCL8. The module and pathway enrichment analyses showed that the common genes had the potential to influence varieties of biological pathways, including the inflammatory response, cytokine activity, cytokine-cytokine receptor interaction, Toll-like receptor signaling pathway, which play an important role in the pathogenesis of hepatitis C and psoriasis. CONCLUSION: Patients with psoriasis display increased prevalence of hepatitis C and the basic related mechanisms between hepatitis C and psoriasis had been preliminarily clarified.

Dysregulated Expression of Arterial MicroRNAs and Their Target Gene Networks in Temporal Arteries of Treatment-Naïve Patients with Giant Cell Arteritis.

In this study, we explored expression of microRNA (miR), miR-target genes and matrix remodelling molecules in temporal artery biopsies (TABs) from treatment-naïve patients with giant cell arteritis (GCA, n = 41) and integrated these analyses with clinical, laboratory, ultrasound and histological manifestations of GCA. NonGCA patients (n = 4) served as controls. GCA TABs exhibited deregulated expression of several miRs (miR-21-5p, -145-5p, -146a-5p, -146b-5p, -155-5p, 424-3p, -424-5p, -503-5p), putative miR-target genes (YAP1, PELI1, FGF2, VEGFA, KLF4) and matrix remodelling factors (MMP2, MMP9, TIMP1, TIPM2) with key roles in Toll-like receptor signaling, mechanotransduction and extracellular matrix biology. MiR-424-3p, -503-5p, KLF4, PELI1 and YAP1 were identified as new deregulated molecular factors in GCA TABs. Quantities of miR-146a-5p, YAP1, PELI1, FGF2, TIMP2 and MMP9 were particularly high in histologically positive GCA TABs with occluded temporal artery lumen. MiR-424-5p expression in TABs and the presence of facial or carotid arteritis on ultrasound were associated with vision disturbances in GCA patients. Correlative analysis of miR-mRNA quantities demonstrated a highly interrelated expression network of deregulated miRs and mRNAs in temporal arteries and identified KLF4 as a candidate target gene of deregulated miR-21-5p, -146a-5p and -155-5p network in GCA TABs. Meanwhile, arterial miR and mRNA expression did not correlate with constitutive symptoms and signs of GCA, elevated markers of systemic inflammation nor sonographic characteristics of GCA. Our study provides new insights into GCA pathophysiology and uncovers new candidate biomarkers of vision impairment in GCA.

Comparison of brain connectomes by MRI and genomics and its implication in Alzheimer's disease.

BACKGROUND: The human brain is complex and interconnected structurally. Brain connectome change is associated with Alzheimer's disease (AD) and other neurodegenerative diseases. Genetics and genomics studies have identified molecular changes in AD; however, the results are often limited to isolated brain regions and are difficult to interpret its findings in respect to brain connectome. The mechanisms of how one brain region impacts the molecular pathways in other regions have not been systematically studied. And how the brain regions susceptible to AD pathology interact with each other at the transcriptome level and how these interactions relate to brain connectome change are unclear. METHODS: Here, we compared structural brain connectomes defined by probabilistic tracts using diffusion magnetic resonance imaging data in Alzheimer's Disease Neuroimaging Initiative database and a brain transcriptome dataset covering 17 brain regions. RESULTS: We observed that the changes in diffusion measures associated with AD diagnosis status and the associations were replicated in an independent cohort. The result suggests that disease associated white matter changes are focal. Analysis of the brain connectome by genomic data, tissue-tissue transcriptional synchronization between 17 brain regions, indicates that the regions connected by AD-associated tracts were likely connected at the transcriptome level with high number of tissue-to-tissue correlated (TTC) gene pairs (P = 0.03). And genes involved in TTC gene pairs between white matter tract connected brain regions were enriched in signaling pathways (P = 6.08 × 10-9). Further pathway interaction analysis identified ionotropic glutamate receptor pathway and Toll receptor signaling pathways to be important for tissue-tissue synchronization at the transcriptome level. Transcript profile entailing Toll receptor signaling in the blood was significantly associated with diffusion properties of white matter tracts, notable association between fractional anisotropy and bilateral cingulum angular bundles (Ppermutation = 1.0 × 10-2 and 4.9 × 10-4 for left and right respectively). CONCLUSIONS: In summary, our study suggests that brain connectomes defined by MRI and transcriptome data overlap with each other.

Glycyrrhizin inactivates toll-like receptor (TLR) signaling pathway to reduce lipopolysaccharide-induced acute lung injury by inhibiting TLR2.

OBJECTIVE: This study aimed to explore glycyrrhizin on acute lung injury (ALI) and how glycyrrhizin (GL) attenuated lipopolysaccharide (LPS)-induced ALI. METHODS: Bioinformatics analysis was performed to screen the expressed genes in LPS-induced ALI mice. The enrichment of functions and signaling pathways of deregulated genes were conducted. Combined with DIGSEE and STICH, the target gene for further investigation was chosen. To verify target gene in mice, we performed experiment in vivo. Forty mice were randomized into NC, LPS, LPS + S, and LPS + GL group. Mice in the LPS + GL group received glycyrrhizin l mg and mice in LPS + S received saline. Then, HE and Masson staining detected pathological changes of lung tissues; enzyme-linked immunosorbent assay analyzed bronchoalveolar lavage fluid concentrations of MIP-2, mice growth-related oncogene homologue (KC), IL-4, IL-6, GM-CSF, IFN-γ, and IgM; western blot analysis determined the expression of toll-like receptor (TLR) signaling and NF-κB pathway-related proteins. RESULTS: Tlr2 which was not only upregulated but also closely related to glycyrrhizin. TLR2 was upregulated in following LPS induced in cells and TLR2 overexpression-activated TLR signaling pathway to promote ALI. After glycyrrhizin treatment, the expression of TLR2 was reduced. Furthermore, it was found out that the number of inflammatory cells, collagen deposition, MIP-2, KC, IL-4, IL-6, GM-CSF, and IFN-γ expression increased in ALI mice and glycyrrhizin mitigated it. Similarly, the expression of TLR signaling pathway and NF-κB pathway-related protein also increased. CONCLUSION: Glycyrrhizin functioned as a suppressor in TLR signaling pathway to reduce LPS-induced ALI by inhibiting TLR2.

RGS1 silencing inhibits the inflammatory response and angiogenesis in rheumatoid arthritis rats through the inactivation of Toll-like receptor signaling pathway.

Emerging evidence shows that rheumatoid arthritis (RA) progression can be induced by the activation of Toll-like receptor (TLR) signaling pathway. Regulator of G-protein signaling 1 (RGS1) is observed to be a candidate biomarker for arthritis. Accordingly, the present study aims to determine the potential effects of RGS1 mediating TLR on RA. A rat model of collagen-induced arthritis (CIA) was established to mimic the features of RA by injection of bovine type II collagen. The rats with CIA were treated with short hairpin RNA (shRNA) against RGS1 or TLR pathway activator Poly I:C to elucidate the role of RGS1 in RA progression. The inflammatory factors were measured, and the thoracic gland and spleen indexes as well as the vascular density were determined. The expression levels of RGS1, TLR3, vascular endothelial growth factor (VEGF), metalloproteinase-2 (MMP-2), MMP-9, and interleukin 1 receptor-associated kinase-4 (IRAK4) were determined. RGS1 was robustly increased in RA. The TLR signaling pathway was suppressed by RGS1 silencing. shRNA-mediated depletion of RGS1 was shown to significantly enhance thoracic gland index and inhibit the serum levels of TNF-α, IL-1ß, and IL-17, spleen index, vascular density, and the expression levels of TLR3, VEGF, MMP-2, MMP-9, and IRAK4. However, when the rats with CIA were treated with Poly I:C, the trend of effects was opposite. These findings highlight that functional suppression of RGS1 inhibits the inflammatory response and angiogenesis by inactivating the TLR signaling pathway in rats with CIA, thereby providing a novel therapeutic target for RA treatment.

Monophosphoryl lipid A induces protection against LPS in medullary thick ascending limb through induction of Tollip and negative regulation of IRAK-1.

LPS inhibits HCO3- absorption in the medullary thick ascending limb (MTAL) through a Toll-like receptor 4 (TLR4)-myeloid differentiation factor 88 (MyD88)-extracellular signal-regulated kinase (ERK) pathway that is upregulated by sepsis. Pretreatment with the nontoxic immunomodulator monophosphoryl lipid A (MPLA) prevents inhibition by LPS through activation of a TLR4-TIR-domain-containing adaptor-inducing interferon-ß (TRIF)-phosphatidylinositol 3-kinase (PI3K) pathway that prevents LPS-induced ERK activation. Here, we identified the molecular mechanisms that underlie the protective inhibitory interaction between the MPLA-PI3K and LPS-ERK pathways. Treatment of mouse MTALs with LPS in vitro increased phosphorylation of IL-1 receptor-associated kinase (IRAK)-1, a critical mediator of LPS signaling downstream of TLR4-MyD88. Activation of ERK by LPS was eliminated by a selective IRAK-1 inhibitor, establishing IRAK-1 as the upstream mediator of ERK activation. Pretreatment of MTALs with MPLA in vitro prevented LPS-induced IRAK-1 activation; this effect was dependent on PI3K. Treatment of MTALs with MPLA increased expression of Toll-interacting protein (Tollip), an inducible protein that negatively regulates LPS signaling by inhibiting IRAK-1. The MPLA-induced increase in Tollip protein level was prevented by PI3K inhibitors. In coimmunoprecipitation experiments, MPLA increased the amount of Tollip stably bound to IRAK-1, an interaction that inhibits IRAK-1 activation. These results support a mechanism whereby MPLA increases Tollip expression in the MTAL through a PI3K-dependent pathway. Tollip, in turn, inhibits LPS-induced TLR4 signaling by suppressing activation of IRAK-1, thereby preventing activation of ERK that inhibits HCO3- absorption. These studies show that MPLA induces reprogramming of MTAL cells that protects against LPS stimulation and identify IRAK-1 and Tollip as new therapeutic targets to prevent renal tubule dysfunction in response to infectious and inflammatory stimuli.

Multiomics analyses reveal that NOD-like signaling pathway plays an important role against Streptococcus agalactiae in the spleen of tilapia.

Streptococcus aglactiae(GBS) infection in tilapia is a serious global disease that causes significant production loss. Here, we studied the role of GBS in the spleen and the spleen's response against the pathogen through dual RNA-seq and proteome technology. Animals were divided into three groups: control, virulent treated (HN016), and attenuated treated (YM001). Spleen samples were collected and analysis when a disease outbreak. Dual RNA-seq result showed the virulence factor genes of GBS, included CAMP factor, PGK, OCT, enolase, scpB, Sip, bca, were upregulation. downregulation of GapA, cylE, OCT, scpB, C5AP, rlmB, hly, FBP, in HN016 and YM001. But for proteomic, OCT and bca were downregulation, the others were upregulation. For host transcriptome KEGG analysis showed, the NOD-like receptor signaling pathway (NLRs) and TOLL-like receptor signaling pathway (TLRs) were upreguoation in HN016 infected fish than the control fish; But for proteome KEGG, only the NLRS was up, the TLRS was not change. Compared with YM001 infected fishes, for transcriptome, NLRs and TLRs in infected HN016 fishes were significance rise (p < 0.01); for proteome, the NLRs was up (p < 0.05), but TLRs was no change.Analysis of pathogen-host interaction showed that the peptidoglycan (PNG), CD2, LCK, and host's Zap70 were involved in the regulation of NLRs; PNG, LCK, and ZAP70 were involved in the regulation of TRLs. Conclusion: the virulent strain HN016 and attenuated strainYM001 differed in the quantity of virulence factors. In tilapia's innate immune system, NLRs was the main defense factors, but bacteria avoided the host defense through TLRs.

Molecular characterization, expression patterns, and functional analysis of toll-interacting protein (Tollip) in Japanese eel Anguilla japonica.

Toll-interacting protein (Tollip) is a key negative regulator of TLR-mediated innate immune responses. The structure and function of Tollip have been well identified in mammals, but the information about Tollip is still limited in teleost fishes. In the present study, the homologue of Tollip was cloned from Japanese eel. It contained an open reading frame encoding a polypeptide of 276 amino acids which shared high identities with other homologues from different species. Multiple alignment of the amino acid sequence showed that the AjTollip protein has the typical conserved domains including an N-terminal Target of Myb1 (Tom1) binding domain (TBD), a central conserved 2 (C2) domain, and a C-terminal coupling of ubiquitin to endoplasmic reticulum degradation (CUE) domain. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed a broad expression for AjTollip in a wide range of tissues, with the highest expression in the liver, a relatively high expression in the spleen, kidney, gills, skin and intestine, and a low expression in the heart and muscle. The AjTollip expressions in the liver and kidney were significantly induced following injection with the bacterial mimic LPS, the viral mimic poly I:C, and Aeromonas hydrophila infection. In vitro, the AjTollip transcripts of Japanese eel liver cells were significantly enhanced by the treatment of LPS, poly I:C, CpG-DNA, and PGN or the stimulation of high concentration of Aeromonas hydrophila (1 × 107 cfu/mL and 1 × 108 cfu/mL). Subcellular localization study showed that AjTollip was mainly distributed in the cytoplasm in a condensed state. When AjTollip was co-transfected with AjMyD88 into HEK293 cells, the luciferase activities of NF-κB were significantly decreased compared with that of AjMyD88 single-transfection groups in natural state or under the stimulation of LPS and poly I:C. These results collectively suggested that AjTollip functions as a negative regulator of MyD88-dependent TLR signaling and plays an important role in fish defense against viral and bacterial infections.

Abnormal bone architecture in mice expressing MyD88 in cells of the osteoclast lineage.

The adapter protein myeloid differentiation primary response gene 88 (MyD88) links the intracellular domains of interleukin receptors 1 and 18, and most Toll-like receptors (TLRs) to interleukin 1 receptor associated kinase (IRAK) signaling and subsequent NF-κB-mediated transcription. Previous work showed that mice with global deficiency of MyD88 (MyD88-/-) have osteopenic cancellous bone along with a reduction in osteoblastic but also osteoclastic surfaces. To further elucidate the role of MyD88 in bone, we utilized mice with osteoclast-restricted MyD88 expression in bone (MyD88OC). Bones of MyD88OC and wild type (wt) mice were examined by microCT analysis. Mechanical properties of bones were tested by three-point bending, and gene expression measured using quantitative real-time polymerase chain reaction. In MyD88OC mice, no osteopenic traits were observed, however, a drastic reduction in geometric parameters was detected. In trabecular bone a loss of connectivity density (-44%, p less than 0.0001) was measured and in cortical bone Imax (-31%, p less than 0.0001), Imin (-20%, p less than 0.001), J (-26%, p less than 0.0001) were reduced. Mechanical testing showed increased load to failure (77%, p less than 0.01) and decreased deflection at failure (-68%, p less than 0.01) of the femur. On the molecular level, relative gene expression analysis showed a (-29%, p less than 0.01) reduction in receptor activator of nuclear factor κ B ligand (RANKL) and no difference in osteoprotegerin (OPG) or RANK. Further, the bone resorption markers cathepsin K (CTSK) and tartrate-resistant acid phosphatase 5 (TRAP) were unchanged. In contrast, the bone formation markers collagen type 1 (COL1A1) and osteocalcin (OC) were decreased by -72% (p less than 0.0001) and -82% (p less than 0.0001), respectively. Together, our data suggests that the function of MyD88 in osteoclasts is sufficient to maintain bone mass, while it fails to preserve bone geometry, likely through dysfunctions in osteoblasts.

CgNrdp1, a conserved negative regulating factor of MyD88-dependent Toll like receptor signaling in oyster Crassostrea gigas.

Toll like receptor (TLR) signaling cascades are under precise regulations to ensure the proper immune responses during various pathogen invasions. The neuregulin receptor degradation protein-1 (Nrdp1) has been demonstrated to be a novel negative regulator of TLR signaling by targeting MyD88 to induce degradation in mammals. In the present study, an Nrdp1 homologue, CgNrdp1, was identified from the genome of Pacific oyster Crassostrea gigas. It contained an open reading frame encoding a polypeptide of 315 amino acids which shared high identities with other homologues from different species. There was a conserved RING domain in CgNrdp1, indicating the functional E3 ubiquitin ligase activity. The bacterially expressed recombinant CgNrdp1 and CgMyD88 showed much stronger affinity compared to control groups in the ELISA assay, showing the interacting ability between CgNrdp1 and CgMyD88. When CgMyD88 or HsMyD88 was co-transfected with CgNrdp1 into HEK293T cells, the luciferase activities of NF-κB were significantly decreased compared to those in MyD88 single-transfection groups, indicating the conserved negative regulating function of CgNrdp1 on the MyD88 induced TLR signaling. These results indicated that CgNrdp1 was a negative regulator of TLR signaling in oyster and the Nrdp1-MyD88 axis was functional and highly conserved from mollusks to mammals in the negative regulation of TLR signaling.

Activation of natural killer T cells contributes to triptolide-induced liver injury in mice.

Triptolide (TP) is the main active ingredient of Tripterygium wilfordii Hook.f, which has attracted great interest due to its promising efficacy for autoimmune diseases and tumors. However, severe adverse reactions, especially hepatotoxicity, have restricted its approval in the market. In the present study we explored the role of hepatic natural killer T (NKT) cells in the pathogenesis of TP-induced liver injury in mice. TP (600 µg/kg/day, i.g.) was administered to female mice for 1, 3, or 5 days. We found that administration of TP dose-dependently induced hepatotoxicity, evidenced by the body weight reduction, elevated serum ALT and AST levels, as well as significant histopathological changes in the livers. However, the mice were resistant to the development of TP-induced liver injury when their NKT cells were depleted by injection of anti-NK1.1 mAb (200 µg, i.p.) on days -2 and -1 before TP administration. We further revealed that TP administration activated NKT cells, dominantly releasing Th1 cytokine IFN-γ, recruiting neutrophils and macrophages, and leading to liver damage. After anti-NK1.1 injection, however, the mice mainly secreted Th2 cytokine IL-4 in the livers and exhibited a significantly lower percentage of hepatic infiltrating neutrophils and macrophages upon TP challenge. The activation of NKT cells was associated with the upregulation of Toll-like receptor (TLR) signaling pathway. Collectively, these results demonstrate a novel role of NKT cells contributing to the mechanisms of TP-induced liver injury. More importantly, the regulation of NKT cells may promote effective measures that control drug-induced liver injury.

Peripheral-blood gene expression profiling studies for coronary artery disease and its severity in Xinjiang population in China.

BACKGROUND: Alterations in gene expression in peripheral blood cells play a curtail role in the presence and extent of coronary artery disease (CAD), but its severity reflected by gene expression alterations in peripheral blood cells is still unknown in Xinjiang population in China. METHODS: Global gene expression profiling in peripheral blood was used to explore differentially expressed genes in coronary artery stenosis patients. RNA was extracted from peripheral blood of 9 controls without coronary stenosis and 21 cases with angiographically CAD. The extent of CAD severity was categorized angiographically as no CAD, mild CAD (20 to 50% luminal diameter stenosis [LDS]), moderate CAD (50 to 75% LDS) and severe CAD (≥75% LDS). Differentially expressed genes related with CAD severity from peripheral blood cells were screened by linear mixed effects analysis using the lme4 package in R. Then the differentially expressed genes that gradually up-regulated or down-regulated were enriched by Gene Ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. RESULTS: The most significantly enrichments were toll-like receptor signaling pathway, immune responses, translational processes, cellular growth, inflammation and metabolic processes. Combined with NCBI-GeneRIF and PubMed analysis, we focused on the 12 genes associated with toll-like receptor signaling pathway in the extent of coronary artery stenosis patients. Receiver operating characteristic (ROC) analysis of 12 genes associated with toll-receptor signaling pathway in the 236 CAD patients from GEO database demonstrated that 12 genes expression could predict severe CAD with an area under the curve of 0.67, sensitivity of 77.65% and specificity of 51.52%. CONCLUSION: These results suggest that 12 genes associated with toll-like receptor signaling pathway in peripheral-blood cells reflect the presence and extent of CAD severity in Xinjiang population in China.