Search

Submitted by azevedo@ufp.pt (azevedo@ufp.pt) on 2018-12-06T17:27:54Z No. of bitstreams: 1 PPG_32814.pdf: 1308383 bytes, checksum: 4f698414bd912c927ae933d260f5360e (MD5) Approved for entry into archive by halves@ufp.pt (halves@ufp.pt) on 2018-12-07T08:10:15Z (GMT) No. of bitstreams: 1 PPG_32814.pdf: 1308383 bytes, checksum: 4f698414bd912c927ae933d260f5360e (MD5) Made available in DSpace on 2018-12-07T08:10:15Z (GMT). No. of bitstreams: 1 PPG_32814.pdf: 1308383 bytes, checksum: 4f698414bd912c927ae933d260f5360e (MD5) Previous issue date: 2018-06-20

Background Tollip, a well‐established endogenous modulator of Toll‐like receptor signaling, is involved in cardiovascular diseases. The aim of this study was to investigate the role of Tollip in neointima formation and its associated mechanisms. Methods and Results In this study, transient increases in Tollip expression were observed in platelet‐derived growth factor‐ BB –treated vascular smooth muscle cells and following vascular injury in mice. We then applied loss‐of‐function and gain‐of‐function approaches to elucidate the effects of Tollip on neointima formation. While exaggerated neointima formation was observed in Tollip‐deficient murine neointima formation models, Tollip overexpression alleviated vascular injury–induced neointima formation by preventing vascular smooth muscle cell proliferation, dedifferentiation, and migration. Mechanistically, we demonstrated that Tollip overexpression may exert a protective role in the vasculature by suppressing Akt‐dependent signaling, which was further confirmed in rescue experiments using the Akt‐specific inhibitor ( AKTI ). Conclusions Our findings indicate that Tollip protects against neointima formation by negatively regulating vascular smooth muscle cell proliferation, dedifferentiation, and migration in an Akt‐dependent manner. Upregulation of Tollip may be a promising strategy for treating vascular remodeling–related diseases.

Toll-interacting protein (Tollip) deficiency has been implicated in complex inflammatory and infectious diseases whose mechanisms are poorly understood. Comparing the gene expression profiles of WT and Tollip-deficient murine embryonic fibroblasts, we observed here that Tollip deficiency selectively reduces the expression of the inflammatory cytokines interleukin 6 (IL-6), IL-12, and tumor necrosis factor α (TNFα) but potentiates the expression of fatty acid–binding protein 4 (FABP4) in these cells. We also observed that expression of hypoxia-inducible factor 1-α (HIF1α) is reduced, whereas that of signal transducer and activator of transcription 5 (STAT5) is elevated, in Tollip-deficient cells, correlating with the decreased expression of inflammatory cytokines and increased expression of FABP4 in these cells. We further found that the coupling of ubiquitin to ER degradation (CUE) domain of Tollip is required for stimulating HIF1α activity, because Tollip CUE–domain mutant cells exhibited reduced levels of HIF1α and selected cytokines. Tollip is known to mediate autophagy and lysosome fusion, and herein we observed that Tollip's autophagy function is required for modulating STAT5 and FABP4 expression. Bafilomycin A, an inhibitor of lysosome fusion, enhanced STAT5 and FABP4 expression in WT fibroblasts, whereas torin 2, an activator of autophagy, reduced STAT5 and FABP4 expression in Tollip-deficient fibroblasts. Taken together, our study reveals that Tollip differentially modulates HIF1α and STAT5 expression in fibroblasts, potentially explaining the complex and context-dependent contribution of Tollip to disease development.

There is increasing evidence pointing toward the role of inflammatory processes in epileptic seizures, and reciprocally, prolonged seizures induce more inflammation in the brain. In this regard, effective strategies to control epilepsy resulting from neuroinflammation could be targeted. Based on the available data, preconditioning (PC) with low dose lipopolysaccharide (LPS) through the regulation of the TLR4 signaling pathway provides neuroprotection against subsequent challenge with injury in the brain. To test this, we examined the effects of a single and chronic brain LPS PC, which is expected to lead to reduction of inflammation against epileptic seizures induced by electroconvulsive shock (ECS). A total of 60 male Sprague Dawley rats were randomly assigned to five groups: control, vehicle (single and chronic), and LPS PC (single and chronic). We first recorded the data regarding the behavioral and histological changes. We further investigated the alterations of gene and protein expression of important mediators in relation to TLR4 and inflammatory signaling pathways. Interestingly, significant increased presence of NFκB inhibitors [Src homology 2-containing inositol phosphatase-1 (SHIP1) and Toll interacting protein (TOLLIP)] was observed in LPS-preconditioned animals. This result was also associated with over-expression of IRF3 activity and anti-inflammatory markers, along with down-regulation of pro-inflammatory mediators. Summarizing, the analysis revealed that PC with LPS prior to seizure induction may have a neuroprotective effect possibly by reprogramming the signaling response to injury.

Bacillus Calmette-Guérin (BCG) immunotherapy is the standard therapy for non-muscle invasive bladder cancer. The aim of the present study was to identify genes that are induced in response to BCG immunotherapy, as these may be potential biomarkers for the response to clinical therapy. To model clinical therapy, human bladder cancer cell lines were incubated with BCG (live or lyophilized BCG Connaught) for 2 h. RNA was extracted and evaluated by Representational Differential Analysis (RDA) and oligo arrays. Gene expression was confirmed by reverse transcription polymerase chain reaction on fresh cell lines with differential abilities to internalize BCG. The effect of 2 major BCG soluble proteins, antigen 85B (Ag85B) and Mycobacterium protein tyrosine phosphatase A (MptpA) and BCG Tice® on gene expression was also determined. GAPDH and β-actin, which are normally used as control genes, were upregulated by BCG. Therefore, the ribosomal RNA gene ribosomal protein S27a was used to normalize gene expression. The genes likely to be induced by BCG internalization and soluble factors were: GSTT2, MGST2, CCL20, TNFα, CCNE1 and IL10RB. Those induced by BCG membrane interactions and/or soluble factors were: MGST1, CXCL6, IL12A, CSF2, IL1β and TOLLIP. MptpA decreased GSTT2 expression, and Ag85B increased TNFα expression. The two BCG strains significantly increased GSTT2, TNFα and TOLLIP levels in MGH cells. However, in J82 cells there was a BCG strain-dependent difference in TNFα expression. An important outcome of the present study was the determination that neither GAPDH nor β-actin were suitable control genes for the analysis of BCG-induced gene expression. BCG Connaught and Tice® induced similar expression levels of genes in bladder cancer cell lines. BCG soluble proteins modulated gene expression and therefore may affect therapeutic outcomes. The genes identified may be novel biomarkers of the response to BCG therapy.

Introduction: Inhibition of Toll-like receptors (TLRs) signaling plays a crucial role in suppressing the inflammation and available data presenting G2013 as an immunomodulatory agent, therefore, we designed this study to answer whether G2013 can affect the signaling pathway of TLR2 and TLR4. Methods: Cytotoxicity study of G2013 was performed by MTT assay. HEK293 TLR2 and HEK293 TLR4 cell lines were cultured and treated with low dose (5µg/ml) and high dose (25µg/ml) of G2013 for 24 hours. Gene expressions of MyD88, Tollip, and NF-κB were defined by quantitative real-time PCR. Results: The cytotoxicity assay showed that the concentrations lesser than 125μg/ml of G3012 had no apparent cytotoxicity, however, the concentrations of 5µg/ml and 25µg/ml could suppress the mRNA expression of MyD88, Tollip and NF-κB in HEK293 TLR2 and HEK293 TLR4 cell lines. Conclusion: in our study, we verified the linkage between the immunosuppressive property of G2013 and TLR2, TLR4 signaling cascade; but so far, the specific target of G2013 and its molecular mechanism has not been detected yet. We recommend further studies on other Patten Recognition Receptors (PRRs)and other mechanisms of inflammation like oxidative stress to be conducted in the future.

Objective: Changes in the brain’s inflammatory status can lead to psychopathological responses, especially depression. Using animal models, recent studies have revealed that this pathology is due, in part, to innate immune responses of monocytes. Methods: We focus on the involvement of Toll-like receptors (TLRs) and expression of genes encoding their negative regulators, SOCS1, TOLLIP, SIGIRR, MyD88s, NOD2, and TNFAIP3, in CD14+ monocytes from 34 patients with major depressive disorder (MDD) and 33 healthy controls before and after treatment with antidepressants. We also seek to investigate their association with depression severity, measured by the 17-item Hamilton Depression Rating Scale (HAMD-17). Results: mRNA expression of all TLRs, except TLR3 and -5, was significantly higher in monocytes from patients with MDD than in those from controls. Conversely, the “brakes” in TLR signaling, including TOLLIP, MyD88s, NOD2, and TNFAIP3, were downregulated. In clinical findings, the remission group showed higher baseline TLR4 and lower baseline IRAK3 mRNA levels but only baseline elevated SOCS1 mRNA levels, which were inversely correlated with HAMD-17 scores, predicting worsened outcome in MDD patients. In addition, TNFAIP3 mRNA levels were increased by antidepressant treatment. Conclusion: Collectively, our findings suggest a role for dysregulation of TLR signaling in monocytes in MDD and identify a balancing effect of antidepressants on this dysregulation.

We studied the peculiarities of the expression of TLR4 and its inhibitor Tollip in placentas obtained from women aged 23-40 years with early- and late-onset preeclampsia. Histological examination of placental tissue (hematoxylin and eosin staining) and immunohistochemical analysis with primary monoclonal antibodies to TLR4 and Tollip were performed on serial paraffin sections. It was found that the expression of TLR4 increased with increasing gestation term in both the syncytiotrophoblast and vascular endothelium of the placental villi (p

Background Toll-like receptor (TLR) -4 mRNA expression is higher in the acute stage of major depressive disorder (MDD) compared with healthy controls. Some previous studies showed that higher TLR-4 expression in male than in female. However, the result is opposite to the higher prevalence of MDD in female. Here, we sought to explore the biological sex difference in mRNA expression levels of negative regulators of the TLR-4 pathway to provide some reasonable explanations. Methods We obtained peripheral blood mononuclear cells (PBMCs) from 100 patients with MDD and 53 healthy controls and used quantitative reverse transcription-polymerase chain reaction analysis for negative regulators of TLR 4 signaling studies. For androgen effect on the TLR regulator expression, we used the embryonic mouse hippocampal cell line (mHippoE-14), dihydrotestosterone as androgen receptor agonist, and flutamide as an antagonist. Results Among healthy controls, PBMCs from males had significantly higher TOLLIP and NOD2 mRNA levels than those from females. By contrast, mRNA for TOLLIP, but not NOD2, was higher in males than females in MDD. In addition, NOD2 and AR mRNAs were found to be lower in male MDD patients than in male healthy controls. Experiments using mHippoE-14 showed that inhibition of AR signaling with the antagonist flutamide suppressed NOD2 expression, whereas treatment AR signaling with the agonist dihydrotestosterone and antagonist flutamide could not increase NOD2 expression. Conclusions TOLLIP and NOD2, negative regulators for TLR-4 pathway, are significantly higher in male than female in health control. The difference in NOD2 disappeared in patients with MDD. The decrease of NOD2 mRNA expression is associated with androgen receptor.

Idiopathic pulmonary fibrosis (IPF) is a devastating lung disease of unknown etiology. The genes TOLLIP and MUC5B play important roles in lung host defense, which is an immune process influenced by oxidative signaling. Whether polymorphisms in TOLLIP and MUC5B modify the effect of immunosuppressive and antioxidant therapy in individuals with IPF is unknown.To determine whether single-nucleotide polymorphisms (SNPs) within TOLLIP and MUC5B modify the effect of interventions in subjects participating in the Evaluating the Effectiveness of Prednisone, Azathioprine, and N-Acetylcysteine in Patients with Idiopathic Pulmonary Fibrosis (PANTHER-IPF) clinical trial.SNPs within TOLLIP (rs5743890/rs5743894/rs5743854/rs3750920) and MUC5B (rs35705950) were genotyped. Interaction modeling was conducted with multivariable Cox regression followed by genotype-stratified survival analysis using a composite endpoint of death, transplantation, hospitalization, or a decline of ≥ 10% in FVC.Significant interaction was observed between N-acetylcysteine (NAC) therapy and rs3750920 within TOLLIP (P interaction = 0.001). After stratifying by rs3750920 genotype, NAC therapy was associated with a significant reduction in composite endpoint risk (hazard ratio, 0.14; 95% confidence interval, 0.02-0.83; P = 0.03) in those with a TT genotype, but a nonsignificant increase in composite endpoint risk (hazard ratio, 3.23; 95% confidence interval, 0.79-13.16; P = 0.10) was seen in those with a CC genotype. These findings were then replicated in an independent IPF cohort.NAC may be an efficacious therapy for individuals with IPF with an rs3750920 (TOLLIP) TT genotype, but it was associated with a trend toward harm in those with a CC genotype. A genotype-stratified prospective clinical trial should be conducted before any recommendation regarding the use of off-label NAC to treat IPF.

TLR-ligands are frequently chosen as candidates for vaccine or adjuvant development because they can primarily bridge innate signaling with adaptive immune responses. Since the adjuvant action of porin, the major outer membrane protein commonly present on Gram-negative bacteria, has been tested on several antigen-presenting cells, we investigated its role in driving systemic immunity which is considered a benchmark for a successful adjuvant. Here, we show porin differentially regulated splenic marginal zone (MZ) and follicular zone (FO) B cell responses in contrast to other classical TLR2-ligands FSL-1 and Pam3CSK4. The protein up-regulated TLR2 and TLR6 and stimulated the activation and costimulatory molecules on FO B cells skewing the cells toward TLR-dependent type-1 cytokine response. However, porin could not up-regulate the TLRs and activate MZ B cells. These cells responded to porin by expressing toll-interacting protein (TOLLIP), the TLR2 and -4 signaling inhibitor along with stimulation of the intracellular pathogen recognition receptor NLR caspase recruitment domain containing protein 5 (NLRC5). The CD1dhi MZ B cells released IL-10 unequivocally demonstrating regulatory B cell feature. Immunization with porin also resulted in transient IL-10 expression by the CD19+CD21hi B cells prior to plasma cell formation. Moreover, the plasma cells developed from the B-2 cell subsets show marked variation in generation of immunoglobulin subclasses. The work delineates multi-faceted role of B cell subsets induced by porin for robust immunity without compromising with the checks and controls.

SummaryEarly endosomes represent the first sorting station for vesicular ubiquitylated cargo. Tollip, through its C2 domain, associates with endosomal phosphatidylinositol 3-phosphate (PtdIns(3)P) and binds ubiquitylated cargo in these compartments via its C2 and CUE domains. Tom1, through its GAT domain, is recruited to endosomes by binding to the Tollip Tom1-binding domain (TBD) through an unknown mechanism. Nuclear magnetic resonance data revealed that Tollip TBD is a natively unfolded domain that partially folds at its N terminus when bound to Tom1 GAT through high-affinity hydrophobic contacts. Furthermore, this association abrogates binding of Tollip to PtdIns(3)P by additionally targeting its C2 domain. Tom1 GAT is also able to bind ubiquitin and PtdIns(3)P at overlapping sites, albeit with modest affinity. We propose that association with Tom1 favors the release of Tollip from endosomal membranes, allowing Tollip to commit to cargo trafficking.

Improper signaling of the IL-36 receptor (IL-36R), a member of the IL-1 receptor family, has been associated with various inflammation-associated diseases. However, the requirements for IL-36R signal transduction remain poorly characterized. This work seeks to define the requirements for IL-36R signaling and intracellular trafficking. In the absence of cognate agonists, IL-36R was endocytosed and recycled to the plasma membrane. In the presence of IL-36, IL-36R increased accumulation in LAMP1+ lysosomes. Endocytosis predominantly used a clathrin-mediated pathway, and the accumulation of the IL-36R in lysosomes did not result in increased receptor turnover. The ubiquitin-binding Tollip protein contributed to IL-36R signaling and increased the accumulation of both subunits of the IL-36R.

Toll-like receptor (TLR) signalling plays an important role in regulating cerebral ischaemia-reperfusion (I/R) injury. Toll-interacting protein (Tollip) is an endogenous negative modulator of TLR signalling that is involved in several inflammatory diseases. Our previous study showed that Tollip inhibits overload-induced cardiac remodelling. However, the role of Tollip in neurological disease remains unknown. In the present study, we proposed that Tollip might contribute to the progression of stroke and confirmed this hypothesis. We found that Tollip expression was significantly increased in I/R-challenged brain tissue of humans, mice and rats in vivo and in primary neurons subjected to oxygen and glucose deprivation in vitro, indicating the involvement of Tollip in I/R injury. Next, using genetic approaches, we revealed that Tollip deficiency protects mice against I/R injury by attenuating neuronal apoptosis and inflammation, as demonstrated by the decreased expression of pro-apoptotic and pro-inflammatory genes and the increased expression of anti-apoptotic genes. By contrast, neuron-specific Tollip over-expression exerted the opposite effect. Mechanistically, the detrimental effects of Tollip on neuronal apoptosis and inflammation following I/R injury were largely mediated by the suppression of Akt signalling. Additionally, to further support our findings, a Tollip knockout rat strain was generated via CRISPR-Cas9-mediated gene inactivation. The Tollip-deficient rats were also protected from I/R injury, based on dramatic decreases in neuronal apoptosis and ischaemic inflammation through Akt activation. Taken together, our findings demonstrate that Tollip acts as a novel modulator of I/R injury by promoting neuronal apoptosis and ischaemic inflammation, which are largely mediated by suppression of Akt signalling.

Ubiquitination is one of the most prevalent posttranslational modifications in eukaryotic cells, with functional importance in protein degradation, subcellular localization and signal transduction pathways. Immunoaffinity enrichment coupled with quantitative mass spectrometry enables the in-depth characterization of protein ubiquitination events at the site-specific level. We have applied this strategy to investigate cellular response triggered by two distinct type agents: small molecule inhibitors of the tumor-associated kinases MEK and PI3K or the pro-inflammatory cytokine IL-17. Temporal profiling of protein ubiquitination events across a series of time points covering the biological response permits interrogation of signaling through thousands of quantified proteins, of which only a subset display significant and physiologically meaningful regulation. Distinctive clusters of residues within proteins can display distinct temporal patterns attributable to diverse molecular functions, although the majority of differential ubiquitination appears as a coordinated response across the modifiable residues present within an individual substrate. In cells treated with a combination of MEK and PI3K inhibitors, we found differential ubiquitination of MEK within the first hour after treatment and a series of mitochondria proteins at later time points. In the IL-17 signaling pathway, ubiquitination events on several signaling proteins including HOIL-1 and Tollip were observed. The functional relevance of these putative IL-17 mediators was subsequently validated by knockdown of HOIL-1, HOIP and TOLIP, each of which decreased IL-17-stimulated cytokine production. Together, these data validate proteomic profiling of protein ubiquitination as a viable approach for identifying dynamic signaling components in response to intracellular and extracellular perturbations.

In our recent article Hörmann and coworkers have reported a role for epithelial cell-intrinsic TLR2 signaling for proliferation and renewal of the small intestinal epithelium. In this study, MyD88 and TRIF expression in the small intestine were affected by gut microbiota. Here, we report that in contrast to TLR2 and its co-receptor TLR1, TLR5 transcripts are not changed by presence of gut microbiota nor regulated through TLR2 or TLR4. Similar to TLR2 also TLR5 depends on MyD88 and TRIF adaptors. Our results indicate that TLR adaptor molecules could be determinants of TLR expression in the small intestine.

Background and Purpose Toll-interacting protein (Tollip) is an endogenous inhibitor of toll-like receptors, a superfamily that plays a pivotal role in various pathological conditions, including myocardial infarction (MI). However, the exact role of Tollip in MI remains unknown.

Histone modifications play critical roles in the regulation of gene expression; however, their roles in the regulation of the innate response remain to be fully investigated. Using transcriptome analysis of mouse immature dendritic cells (DCs) and LPS-induced mature DCs, we identified that Ezh1 was the most upregulated histone methyltransferase during DC maturation. In this study, we investigated the role of Ezh1 in regulating the innate immune response. We found that silencing of Ezh1 significantly suppressed TLR-triggered production of cytokines, including IL-6, TNF-α, and IFN-β, in DCs and macrophages. Accordingly, TLR-activated signaling pathways were impaired in Ezh1-silenced macrophages. By transcriptome analysis of Ezh1-silenced macrophages, we found that Toll-interacting protein (Tollip), one well-known negative regulator of TLR signaling, was upregulated. Silencing of Tollip rescued TLR-triggered cytokine production in Ezh1-silenced macrophages. The SET domain of Ezh1 is essential for its enhancing effect on the TLR-triggered innate immune response and downstream signaling, indicating that Ezh1 promotes a TLR-triggered innate response through its lysine methyltransferase activity. Finally, Ezh1 was found to suppress the transcription of Tollip by directly targeting the proximal promoter of tollip and maintaining the high level of trimethylation of histone H3 lysine 27 there. Therefore, Ezh1 promotes TLR-triggered inflammatory cytokine production by suppressing the TLR negative regulator Tollip, contributing to full activation of the innate immune response against invading pathogens.

How composition of egg yolk (EY) influences NF-κB, a key transcription pathway in inflammation, remains unclear. We performed partial delipidation of EY that removed 20-30% of cholesterol and triglycerides. The resulting polar and nonpolar fractions were termed EY-P and EY-NP. NF-κB activation in response to EY from different suppliers and their fractions was examined in 3T3-L1 adipocytes using a NF-κB response element reporter assay and by analyzing expression of 248 inflammatory genes. Although EY-P and EY contained similar level of vitamins, carotenoids, and fatty acids, only delipidated EY-P fraction suppressed NF-κB via down-regulation of toll like receptor-2 and up-regulation of inhibitory toll interacting protein (Tollip) and lymphocyte antigen 96 (Ly96). Our data suggest that anti-inflammatory activity of lutein and retinol were blunted by nonpolar lipids in EY, likely via crosstalk between SREBP and NF-κB pathways in adipocytes. Thus, moderate delipidation may improve the beneficial properties of regular eggs.

Myeloid differentiation factor 88 (MyD88) and Toll-interacting protein (Tollip) are two important regulatory proteins of the Toll-like receptor (TLR) signaling pathways. In this paper, a Tollip sequence of grouper (Epinephelus coioides) was identified and the signal transduction functions of Tollip and MyD88 were studied. The full length of E. coioides Tollip (EcTollip) cDNA with an open reading frame (ORF) of 1734 nucleotides encoded a putative protein of 274 amino acid residues. The EcTollip protein had conservative domains with mammalian homologous proteins, and high identity (78%–95%) with other vertebrates. MyD88 and Tollip were distributed in the HeLa cytoplasm in a highly condensed form. Over-expression of MyD88 could activate nuclear factor-κB (NF-κB) and its function was dependent on the death domain and ID domain on the N-terminal. Some important functional sites of mammalian MyD88 also affected fish MyD88 signal transduction. Tollip impaired NF-κB signals activated by MyD88, and its activity was dependent on the coupling of ubiquitin to the endoplasmic reticulum degradation (CUE) domain on the C-terminal. These results suggest that MyD88 and Tollip of fish and mammals are conservative on function during evolution.

Toll-interacting protein (Tollip) is a critical regulator of Toll-like receptor (TLR)-mediated innate immune responses. However, the Tollip gene has not been systematically characterized in shellfish. In this study, we identified and characterized a Tollip gene, PyTollip, in Yesso scallop (Patinopecten yessoensis). Phylogenetic and protein structural analyses were conducted to determine its sequence identities and evolutionary relationships. Compared with Tollip genes from other invertebrate and vertebrate species, the PyTollip gene is highly conserved in its sequence and structural features, except that a unique asparagine residue was found at a conserved site in the C2 domain of PyTollip. Quantitative real-time PCR was used to investigate the expression profiles of PyTollip in different developmental stages, healthy adult tissues, and in hemolymph after Micrococcus luteus and Vibrio anguillarum bacterial infection. Real-time PCR analysis demonstrated differential expression of PyTollip at the acute phase (3 h) after infection with Gram-negative (V. anguillarum) and Gram-positive (M. luteus) bacteria. A second strong response of PyTollip expression was observed 24 h after challenge with V. anguillarum. Collectively, these results provide novel insights into the specific role and response of Tollip and TLR signaling pathways in host immune responses against different bacterial pathogens in bivalves.

Idiopathic pulmonary fibrosis (IPF) is a chronic, debilitating, fibrotic lung disease leading to respiratory failure and ultimately to death. Being the prototype of interstitial lung diseases, IPF is characterized by marked heterogeneity regarding its clinical course. Despite significant progress in the understanding of its pathogenesis, we still cannot reliably predict the course of the disease and the response to treatment of an individual patient. Non-invasive biomarkers, in particular serum biomarkers, for the (early) diagnosis, differential diagnosis, prognosis and prediction of therapeutic response are urgently needed. Numerous molecules involved in alveolar epithelial cell injury, fibroproliferation and matrix remodeling as well as immune regulation have been proposed as potential biomarkers. Furthermore, genetic variants of TOLLIP, MUC5B, and other genes are associated with a differential response to treatment and with the development and/or the prognosis of IPF. Additionally, the bacterial signature in IPF lungs, as shown from microbiome analyses, as well as mitochondrial DNA seem to have promising roles as biomarkers. Moreover, combination of multiple biomarkers may identify comprehensive biomarker signatures in IPF patients. However, there is still a long way until these potential biomarkers complete or substitute for the clinical and functional parameters currently available for IPF.

Toll-interacting protein (Tollip) is a key negative regulator of innate immunity by preventing excessive proinflammatory responses. Tollip genetic variation has been associated with airflow limitation in asthma subjects and Tollip expression. Whether Tollip regulates lung inflammation in a type 2 cytokine milieu (e.g., IL-13) is unclear. Our goal was to determine the in vivo role of Tollip in IL-13-mediated lung eosinophilic inflammation and the underlying mechanisms. Tollip-knockout (KO) and wild-type (WT) mice were inoculated intranasally with recombinant mouse IL-13 protein to examine lung inflammation. To determine how Tollip regulates inflammation, alveolar macrophages and bone marrow-derived macrophages from Tollip KO and WT mice were cultured with or without IL-13 and/or IL-33. IL-13-treated Tollip KO mice significantly increased lung eosinophilic inflammation and eotaxin-2 (CCL24) levels compared with the WT mice. IL-13- treated Tollip KO (vs. WT) macrophages, in the absence and particularly in the presence of IL-33, increased expression of the IL-33 receptor ST2L and CCL24, which was in part dependent on enhanced activation of interleukin (IL)-1 receptor-associated kinase 1 (IRAK1) and signal transducer and activator of transcription 6 (STAT6). Our results suggest that Tollip downregulates IL-13-mediated pulmonary eosinophilia in part through inhibiting the activity of the ST2L/IL-33/IRAK1 axis and STAT6.

Susceptibility to malaria infection has been associated with host genetic polymorphisms that differs between groups. We hypothesize that Toll-interacting proteins (TOLLIP), vitamin D receptor (VDR) and tumor necrosis factor-α (TNF) genes are significant contributors to susceptibility and disease severity in Plasmodium falciparum (Pf) infection. Our aim is to explore the genomic diversity and haplotype frequency of these genes, as well as extrapolate possible association with markers of severity, between malaria-infected and healthy controls. Genomic DNA samples extracted from the blood of 107 malaria-infected patients and 190 uninfected controls were analyzed, with no difference in genotypic or allelic frequencies of TOLLIP and VDR polymorphisms. However, a significant difference in the genotypic (p = 2.20E-16) and allelic frequencies (p = 2.20E-16) of the TNF-α (snp rs1800629) polymorphism was found. The preponderance of the mutant variant among the malaria-infected show a possible impaired capacity to mount an effective immune response, potentially confirmed by our association results. This result calls for analysis of clearly delineated uncomplicated versus severe disease groups, including serum assays, providing a basis to conclude that susceptibility to malaria infection and potential contribution to disease severity is significantly associated with polymorphisms of the tumor necrosis factor-α but not TOLLIP or VDR genes.

Pam3CSK4 is a synthetic tripalmitoylated lipopeptide that acts as a ligand of TLR1/TLR2 by mimicking the acetylated amino terminus of bacterial lipoproteins. Here we found that pretreatment of Pam3CSK4 protected mice from systemic infection of methicillin-resistant Staphylococcus aureus (MRSA), and enhanced the bacterial clearance in bacteremia model. Pro-inflammatory cytokines, such as TNF-α, IL-6, MCP-1 and IFN-γ were significantly decreased in serum from Pam3CSK4-treated mice. Besides, upon PamCSK4 treatment, the TLR2 expression was down-regulated, IRAK1 phosphorylation was inhibited, and the expression of IRAK-M and Tollip, two negative regulators of NF-κB pathway, was up-regulated. All of these indicated that Pam3CSK4 attenuated inflammation via inhibiting TLR1/TLR2 and the downstream NF-κB pathways, and suggested that Pam3CSK4 could be a potential immune modulator for MRSA systemic infection.

Leucine-rich repeat kinase 2 (LRRK2) is a Ser/Thr kinase having mixed lineage kinase-like and GTPase domains, controlling neurite outgrowth and neuronal cell death. Evidence suggests that LRRK2 is involved in innate immune response signaling, but the underlying mechanism is yet unknown. A novel protein inhibitor of phosphatase 3B, RCAN1, is known to positively regulate inflammatory signaling through modulation of several intracellular targets of interleukins in immune cells. In the present study, we report that LRRK2 phosphorylates RCAN1 (RCAN1-1S) and is markedly up-regulated during interleukin-1 beta (IL-1 beta) treatment. During IL-1 beta treatment, LRRK2mediated phosphorylation of RCAN1 promoted the formation of protein complexes, including that between Tollip and RCAN1. LRRK2 decreased binding between Tollip and IRAK1, which was accompanied by increased formation of the IRAK1-TRAF6 complex. TAK1 activity was significantly enhanced by LRRK2. Furthermore, LRRK2 enhanced transcriptional activity of NF-kappa B and cytokine IL-8 production. These findings suggest that LRRK2 might be important in positively modulating IL-1 beta-mediated signaling through selective phosphorylation of RCAN1.

Innate leukocytes manifest dynamic and distinct inflammatory responses upon challenges with rising dosages of pathogen-associated molecular pattern molecules such as lipopolysaccharide (LPS). To differentiate signal strengths, innate leukocytes may utilize distinct intracellular signaling circuitries modulated by adaptor molecules. Toll-interacting protein (Tollip) is one of the critical adaptor molecules potentially playing key roles in modulating the dynamic adaptation of innate leukocytes to varying dosages of external stimulants. While Tollip may serve as a negative regulator of nuclear factor κ of activated B cells signaling pathway in cells challenged with higher dosages of LPS, it acts as a positive regulator for low-grade chronic inflammation in leukocytes programmed by subclinical low-dosages of LPS. This review aims to discuss recent progress in our understanding of complex innate leukocyte dynamics and its relevance in the pathogenesis of resolving versus non-resolving chronic inflammatory diseases.

Background Inhibition of Toll-like receptors (TLRs) signaling has been established as a new method for the development of anti-inflammatory drugs instead of NSAIDs (non-steroid anti-inflammatory drugs). Since the immunomodulatory role of G2013 (α-L-Guluronic acid) was reported in some recent experiments, we decided to assess the effects of G2013 on the protein expression of TLR2 and TLR4, their downstream signaling cascade, and the secretion of pro-inflammatory cytokines in human peripheral blood mononuclear cells (PBMCs). Methods After blood sampling from 16 healthy donors, PBMCs were isolated and treated with/without lipopolysaccharide (LPS), lipopolyteichoic acid (LTA), and G2013. Flow cytometry was done for detecting the protein expression of TLR2 and TLR4. MyD88, IκB, Tollip, and NF-κB mRNA expression were assessed by realtime PCR. ELISA was performed for assessing the concentration of IL-1β and IL-6. Results G2013 at a concentration of 25 µg/mL (high dose) significantly downregulated NF-κB, IκB and MyD88 mRNA expression and suppressed the secretion of IL-1β by PBMCs. The findings indicate that G2013 may exert its regulatory effect under normal condition via Tollip in a dose dependence pathway. Our results demonstrated that G2013 had no profound impact on the protein expression of TLR2 and TLR4. Conclusion In conclusion, our findings point to the immunomodulatory effect of G2013 on the TLR2 and TLR4 signaling cascade and cytokine production by PBMCs. These findings could lead to an establishment of new safe anti-inflammatory drugs in the future.

Interferon (IFN)-γ and tumor necrosis factor (TNF)-α cause chronic inflammation of the intestine leading to progression of inflammatory bowel disease (IBD), which is manifested through rapid apoptosis of the intestinal epithelial cells (iECs). Here, we show inhibition of IFN-γ and TNF-α-induced apoptosis of INT-407 cells by porin, a microbe-associated molecular pattern (MAMP) with affinity for toll-like receptor (TLR)2 and commonly present in Gram-negative bacteria. Proinflammatory cytokines induce apoptosis by activation of caspase 8 that triggers caspase 9 through Bax finally leading to activation of caspase 3, the executioner caspase. Interestingly, while IFN-γ and TNF-α promotes Bax expression, in contrast porin up-regulates anti-apoptotic Bcl-xL resulting in iEC survivability. We show elevated expression of TLR2 is a key requisite for IFN-γ and TNF-α mediated caspase 8 up-regulation that contributes to apoptosis of iECs. Down-regulation of TLR2 expression is central for checking apoptosis which is achieved by elevated level of toll-interacting protein (TOLLIP) in presence of porin. Attempts to limit IBD is in progress with anti-IFN-γ and anti-TNF-α Abs or use of IL-10. Although probiotic bacterial proteins have shown to successfully reduce IFN-γ and TNF-α mediated apoptosis, the exact mechanism of their action has remained elusive. This study identifies the underlying sequential events of transient TLR2 stimulation followed by its blocking in response to the bacterial outer membrane protein, which advocates intervention at TLR-juncture is crucial for controlling IBD.

Xuebijing (XBJ) is a type of traditional Tibetan medicine, and previous pharmacological studies have shown that the ethanol extract is derived from Chuanxiong, Chishao, Danshen and Honghua. Chuanxiong, Chishao, Danshen and Honghua possesses potent anti-inflammatory activity, and has been used in the treatment of inflammatory infectious diseases. In the present study, we investigated the effects of XBJ on pulmonary permeability and lung injury in cecal ligation and puncture (CLP)-induced sepsis in rats. A CLP sepsis model was established for the control and treatment groups, respectively. Approximately 2 h prior to surgery, an amount of 100 mg/kg XBJ injection was administered to the treatment group. Reverse transcription polymerase chain reaction (PT-PCR) and western blot analysis were used to examine the expression of Toll-interacting protein (Tollip), interleukin-1 receptor-associated kinase 1 (IRAK1), Toll-like receptor 4 (TLR4), nuclear factor-κB65 (NF-κB65) and TNF receptor-associated factor 6 (TRAF6) in lung tissue. ELISA was applied to detect changes of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1 (IL-1), interleukin-4 (IL-4) and interleukin-10 (IL-10) levels in bronchoalveolar lavage (BAL) fluid, and intercellular adhesion molecule 1 (ICAM-1) and von Willebrand factor (vWF) in serum. The number of neutrophils, albumin and total cells in the BAL fluid were measured. For histological analysis, hematoxylin and eosin (H&E) stains were evaluated. Lung permeability, the wet/dry weight ratio (W/D) and the lung pathology score were determined following the induction of ALI by CLP for 24 h. The results demonstrated that XBJ upregulated Tollip expression and blocked the activity of IRAK1, TLR4, NF-κβ65 and TRAF6. Additionally, the number of neutrophils and total cells were significantly decreased in the XBJ group compared to that in the control group. Lung permeability, the wet/dry weight ratio (W/D) and the lung pathology score were significantly decreased in the XBJ group. The histological results also demonstrated the attenuation effect of XBJ on CLP-induced lung inflammation. The results of the present study indicated that XBJ has a significantly reduced CLP-induced lung permeability by upregulating Tollip expression. The protective effects of XBJ suggest its therapeutic potential in CLP-induced acute lung injury treatment.

The green tea catechin (‐)-epigallocatechin-3-O-gallate (EGCG) is known to exhibit various biological and pharmacological properties. We have identified 67-kDa laminin receptor (67LR) as a cell-surface EGCG receptor that confers EGCG responsiveness to many cancer cells at physiological concentrations. Here we provide an overview of several pathways that sense and manage the activities of EGCG. EGCG has been shown to rescue mice from lipopolysaccharide (LPS)-induced lethal endotoxemia and downregulate inflammatory responses in macrophages. LPS is one of the most powerful activators of toll-like receptor (TLR)4 signaling and is also well known to induce production of inflammatory mediators. A negative regulator of TLR signaling, Toll-interacting protein (Tollip), is indispensable for mediating the anti-inflammatory action of EGCG, and its protein expression level is upregulated by EGCG through 67LR. Additionally, EGCG can reduce the expression of TLR4 via 67LR. Using a direct genetic screen, eukaryotic translation elongation factor 1A (eEF1A) is identified as a component responsible for the anti-melanoma activity of EGCG. EGCG induces the dephosphorylation of myosin phosphatase targeting subunit 1 (MYPT1) at Thr-696 and activates myosin phosphatase through both eEF1A and 67LR. Silencing of 67LR, eEF1A, or MYPT1 in tumor cells results in abrogation of EGCG-induced tumor growth inhibition in vivo. Additionally, we found that eEF1A is up-regulated by EGCG through 67LR. EGCG has been shown to be able to induce apoptotic cell death in multiple myeloma cells through the 67LR, while having no significant effect on peripheral blood mononuclear cells (PBMCs). The expression of 67LR was significantly elevated in myeloma cells compared to PBMCs. We found that the apoptosis-inducing activity of EGCG in multiple myeloma cells is attributable to the activation of acid sphingomyelinase (ASM) that hydrolyzes sphingomyelin into ceramide. EGCG induces ASM translocation to the plasma membrane and protein kinase C delta (PKCδ) phosphorylation at Ser 664 , which was necessary for ASM/ceramide signaling, via 67LR. Furthermore, EGCG activates PKCδ/ASM/ceramide pathway by activating Akt/eNOS/NO/cGMP signaling through 67LR. We also found that the upregulation of cGMP is a rate-determining process of this cell death pathway, and cancer-overexpressed negative regulator of cGMP, PDE5 attenuates the cell death induced by EGCG. Vardenafil, one of the PDE5 selective inhibitors used for treating erectile dysfunction potentiates the anti-cancer effect of EGCG. These results demonstrate that cGMP elevation caused by targeting the overexpressed 67LR and PDE5 in cancer cells may be a useful approach for cancer-specific chemotherapy.

Summary Selective ubiquitin-dependent autophagy plays a pivotal role in the elimination of protein aggregates, assemblies, or organelles and counteracts the cytotoxicity of proteins linked to neurodegenerative diseases. Following substrate ubiquitylation, the cargo is delivered to autophagosomes involving adaptors like human p62 that bind ubiquitin and the autophagosomal ubiquitin-like protein Atg8/LC3; however, whether similar pathways exist in lower eukaryotes remained unclear. Here, we identify by a screen in yeast a new class of ubiquitin-Atg8 adaptors termed CUET proteins, comprising the ubiquitin-binding CUE-domain protein Cue5 from yeast and its human homolog Tollip. Cue5 collaborates with Rsp5 ubiquitin ligase, and the corresponding yeast mutants accumulate aggregation-prone proteins and are vulnerable to polyQ protein expression. Similarly, Tollip depletion causes cytotoxicity toward polyQ proteins, whereas Tollip overexpression clears human cells from Huntington's disease-linked polyQ proteins by autophagy. We thus propose that CUET proteins play a critical and ancient role in autophagic clearance of cytotoxic protein aggregates.

lymphocytes in response to heat-stable enterotoxigenic Escherichia coli (ETEC) pathogen-associated molecular patterns (PAMPs), while the immunoregulatory effect of B. adolescentis ATCC15705 was significantly lower than that observed for the MCC-117 strain. Considering the different capacities of the two bifidobacterium strains to activate toll-like receptor (TLR)-2 and their differential immunoregulatory activities in PIE and immune cells, we hypothesized that comparative studies with both strains could provide important information regarding the molecular mechanism(s) involved in the anti-inflammatory activity of bifidobacteria. In this work, we demonstrated that the anti-inflammatory effect of B. breve MCC-117 was achieved by a complex interaction of multiple negative regulators of TLRs as well as inhibition of multiple signaling pathways. We showed that B. breve MCC-117 reduced heat-stable ETEC PAMP-induced NF-κB, p38 MAPK and PI3 K activation and expression of pro-inflammatory cytokines in PIE cells. In addition, we demonstrated that B. breve MCC-117 may activate TLR2 synergistically and cooperatively with one or more other pattern recognition receptors (PRRs), and that interactions may result in a coordinated sum of signals that induce the upregulation of A20, Bcl-3, Tollip and SIGIRR. Upregulation of these negative regulators could have an important physiological impact on maintaining or reestablishing homeostatic TLR signals in PIE cells. Therefore, in the present study, we gained insight into the molecular mechanisms involved in the immunoregulatory effect of B. breve MCC-117.Key words: bifidobacteria, anti-inflammatory activity, porcine intestinal epithelial cells, Toll-like receptors negative regulators, Toll-like receptor 2INTRODUCTIONIn recent years, great advances have been made with regard to knowledge of the mechanism involved in probiotic effects, especially those related to the capacity to stimulate the immune system [1–3]. In this regard, it was reported that Toll-like receptor (TLR)-2 may play an important regulatory role in the recognition of bifidobacteria that possess an immunoinhibitory effect. Bifidobacteria have been shown to inhibit the production of tumor necrosis factor (TNF)-α and interleukin (IL)-6 induced by immunostimulatory lactobacilli in blood immune cells via interaction with TLR2 [4]. In addition, Hoarau et al. [5] demonstrated that the supernatant of Bifidobacterium breve C50 induces dendritic cell (DCs) maturation through TLR2, with a high level of IL-10 production. A study, using DCs from TLR2−/− mice reported that bifidobacteria induced much higher levels of IL-12 and lower IL-10 levels in DCs from TLR2−/− mice when compared with wild-type DCs [4].Considering these observations, we isolated porcine TLR2 (pTLR2) cDNA from ileal Peyer’s patches (PPs) and, using a human cell line, developed a method for evaluating the immune responses to immunobiotic bifidobacteria by constructing a pTLR2-expressing

Although tumor immune environment is increasingly recognized to be highly important during the modulation of tumorigenesis and tumor regression, the role and regulation of innate leukocytes such as neutrophils during the modulation of tumor immune environment remain controversial and less defined. In this study, we tested the hypothesis that removal of innate signaling suppressors may boost the anti-tumor immune function of innate neutrophils in vitro and in vivo. To this regard, we examined two key innate signaling suppressors Tollip and IRAK-M and their roles in modulating neutrophil anti-tumor immune functions. We observed that selective deletion of Tollip enhanced tumor immune surveillance in the AOM-DSS chemically induced colon cancer model. Tollip deficiency released the neutrophil suppression on T cell proliferation and activation. The adoptive transfer of Tollip deficient neutrophils were sufficient to transfer enhanced tumor immune surveillance and reduce tumor burden. Likewise, the study of another innate suppressor IRAK-M revealed that IRAK-M expression was up-regulated in the human patients with colorectal cancer. We also demonstrated that IRAK-M deficient mice exhibited reduced tumor burden following AOM-DSS challenge. Together, our data reveal a novel anti-tumor immune-enhancement strategy through utilizing reprogrammed neutrophils with targeted removal of innate signaling suppressors. Citation Format: Yao Zhang, Christina Lee, Na Diao, Liwu Li. Removal of innate suppressors facilitates tumor-immune surveillance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1515.

Leukocyte telomere length (LTL), MUC5B rs35705950 and TOLLIP rs5743890 have been associated with idiopathic pulmonary fibrosis (IPF).In this observational cohort study, we assessed the associations between these genomic markers and outcomes of survival and rate of disease progression in patients with interstitial pneumonia with autoimmune features (IPAF, n=250) and connective tissue disease-associated interstitial lung disease (CTD-ILD, n=248). IPF (n=499) was used as a comparator.The LTL of IPAF and CTD-ILD patients (mean age-adjusted log-transformed T/S of −0.05±0.29 and −0.04±0.25, respectively) is longer than that of IPF patients (−0.17±0.32). For IPAF patients, LTL versus −0.86% per year; pMUC5B rs35705950 minor allele frequency (MAF) is greater for IPAF patients (23.2, 95% CI 18.8–28.2; pversus −0.01±0.23; p=0.00055) and higher MUC5B MAF (34.6, 95% CI 24.4–46.3 versus 14.1, 95% CI 9.8–20.0; p=0.00025). Neither LTL nor MUC5B are associated with transplant-free CTD-ILD survival.LTL and MUC5B MAF have different associations with lung function progression and survival for IPAF and CTD-ILD.

Aims Toll-interacting protein (Tollip) is a critical regulator of the Toll-like receptor-mediated signalling pathway. However, the role of Tollip in chronic pressure overload-induced cardiac hypertrophy remains unclear. This study aimed to determine the functional significance of Tollip in the regulation of aortic banding-induced cardiac remodelling and its underlying mechanisms. Methods and results First, we observed that Tollip was down-regulated in human failing hearts and murine hypertrophic hearts, as determined by western blotting and RT–PCR. Using cultured neonatal rat cardiomyocytes, we found that adenovirus vector-mediated overexpression of Tollip limited angiotensin II-induced cell hypertrophy; whereas knockdown of Tollip by shRNA exhibited the opposite effects. We then generated a transgenic (TG) mouse model with cardiac specific-overexpression of Tollip and subjected them to aortic banding (AB) for 8 weeks. When compared with AB-treated wild-type mouse hearts, Tollip-TGs showed a significant attenuation of cardiac hypertrophy, fibrosis, and dysfunction, as measured by echocardiography, immune-staining, and molecular/biochemical analysis. Conversely, a global Tollip-knockout mouse model revealed an aggravated cardiac hypertrophy and accelerated maladaptation to chronic pressure overloading. Mechanistically, we discovered that Tollip interacted with AKT and suppressed its downstream signalling pathway. Pre-activation of AKT in cardiomyocytes largely offset the Tollip-elicited anti-hypertrophic effects. Conclusion Our results provide the first evidence that Tollip serves as a negative regulator of pathological cardiac hypertrophy by blocking the AKT signalling pathway.

The cell type-, organ-, and species-specific expression of the pattern-recognition receptors (PRRs) are well described but little is known about the respective expression profiles of their negative regulators. We therefore determined the mRNA expression levels of A20, CYLD, DUBA, ST2, CD180, SIGIRR, TANK, SOCS1, SOCS3, SHIP, IRAK-M, DOK1, DOK2, SHP1, SHP2, TOLLIP, IRF4, SIKE, NLRX1, ERBIN, CENTB1, and Clec4a2 in human and mouse solid organs. Humans and mice displayed significant differences between their respective mRNA expression patterns of these factors. Additionally, we characterized their expression profiles in mononuclear blood cells upon bacterial endotoxin, which showed a consistent induction of A20, SOCS3, IRAK-M, and Clec4a2 in human and murine cells. Furthermore, we studied the expression pattern in transient kidney ischemia-reperfusion injury versus post-ischemic atrophy and fibrosis in mice. A20, CD180, ST2, SOCS1, SOCS3, SHIP, IRAK-M, DOK1, DOK2, IRF4, CENTB1, and Clec4a2 were all induced, albeit at different times of injury and repair. Progressive fibrosis was associated with a persistent induction of these factors. Thus, the organ- and species-specific expression patterns need to be considered in the design and interpretation of studies related to PRR-mediated innate immunity, which seems to be involved in tissue injury, tissue regeneration and in progressive tissue scarring.

To elucidate the significance of Toll-like receptors and their negative regulating factors PPAR-γ and Tollip on the pathogenesis of colitis. Colitis model was induced by TNBS in rat. The expression of TLR2, TLR4, NF-κBp65, PPAR-γ and Tollip was examined by immunohistochemistry (IHC) and reverse-transcription polymerase chain reaction (RT-PCR). RT-PCR revealed a significant increased expression of TLR2, TLR4, and NF-κBp65 in the colitis group compared with the normal group (TLR2: 1.057 ± 0.092, 0.463 ± 0.101, t = 4.125, P = 0.001; TLR4: 0.376 ± 0.029, 0.215 ± 0.049, t = 2.731, P = 0.013; NF-κBp65: 0.746 ± 0.049, 0.206 ± 0.063, t = 6.055, P = 0.000). The expression was positively correlated with the generally damage score and the histological injury score correspondingly (TLR2: r = 0.573, r = 0.559; TLR4: r = 0.754, r = 0.866; NF-κBp65: r = 0.548, r = 0.919). The Tollip mRNA wasn't obviously diversity between the normal and colitis groups by RT-PCR (Tollip: 0.288 ± 0.050, 0.140 ± 0.046, t = 1.993, P = 0.061). While the Tollip protein was mainly assembled in the lamina propriaand higher in the colitis group compared with the normal group by IHC. The expression of PPAR-γ in the colitis group was obviously lower than that in the normal group (PPAR-γ: 0.255 ± 0.065, 0.568 ± 0.072, t = 2.882, P = 0.010). The expression of Tollip and PPAR-γ was negative correlated with the generally damage score and histological injury score correspondingly (Tollip: r = -0.497, r = -0.551; PPAR-γ: r = -0.683, r = -0.853). The disbalance between TLRs and their negative regulating factors PPAR-γ and Tollip was closely associated with the course of colitis.

Summary Background Idiopathic pulmonary fibrosis (IPF) is a devastating disease that probably involves several genetic loci. Several rare genetic variants and one common single nucleotide polymorphism (SNP) of MUC5B have been associated with the disease. Our aim was to identify additional common variants associated with susceptibility and ultimately mortality in IPF. Methods First, we did a three-stage genome-wide association study (GWAS): stage one was a discovery GWAS; and stages two and three were independent case-control studies. DNA samples from European-American patients with IPF meeting standard criteria were obtained from several US centres for each stage. Data for European-American control individuals for stage one were gathered from the database of genotypes and phenotypes; additional control individuals were recruited at the University of Pittsburgh to increase the number. For controls in stages two and three, we gathered data for additional sex-matched European-American control individuals who had been recruited in another study. DNA samples from patients and from control individuals were genotyped to identify SNPs associated with IPF. SNPs identified in stage one were carried forward to stage two, and those that achieved genome-wide significance (p −8 ) in a meta-analysis were carried forward to stage three. Three case series with follow-up data were selected from stages one and two of the GWAS using samples with follow-up data. Mortality analyses were done in these case series to assess the SNPs associated with IPF that had achieved genome-wide significance in the meta-analysis of stages one and two. Finally, we obtained gene-expression profiling data for lungs of patients with IPF from the Lung Genomics Research Consortium and analysed correlation with SNP genotypes. Findings In stage one of the GWAS (542 patients with IPF, 542 control individuals matched one-by-one to cases by genetic ancestry estimates), we identified 20 loci. Six SNPs reached genome-wide significance in stage two (544 patients, 687 control individuals): three TOLLIP SNPs (rs111521887, rs5743894, rs5743890) and one MUC5B SNP (rs35705950) at 11p15.5; one MDGA2 SNP (rs7144383) at 14q21.3; and one SPPL2C SNP (rs17690703) at 17q21.31. Stage three (324 patients, 702 control individuals) confirmed the associations for all these SNPs, except for rs7144383. Linkage disequilibrium between the MUC5B SNP (rs35705950) and TOLLIP SNPs (rs111521887 [ r 2 =0·07], rs5743894 [ r 2 =0·16], and rs5743890 [ r 2 =0·01]) was low. 683 patients from the GWAS were included in the mortality analysis. Individuals who developed IPF despite having the protective TOLLIP minor allele of rs5743890 carried an increased mortality risk (meta-analysis with fixed-effect model: hazard ratio 1·72 [95% CI 1·24–2·38]; p=0·0012). TOLLIP expression was decreased by 20% in individuals carrying the minor allele of rs5743890 (p=0·097), 40% in those with the minor allele of rs111521887 (p=3·0 × 10 −4 ), and 50% in those with the minor allele of rs5743894 (p=2·93 × 10 −5 ) compared with homozygous carriers of common alleles for these SNPs. Interpretation Novel variants in TOLLIP and SPPL2C are associated with IPF susceptibility. One novel variant of TOLLIP , rs5743890, is also associated with mortality. These associations and the reduced expression of TOLLIP in patients with IPF who carry TOLLIP SNPs emphasise the importance of this gene in the disease. Funding National Institutes of Health; National Heart, Lung, and Blood Institute; Pulmonary Fibrosis Foundation; Coalition for Pulmonary Fibrosis; and Instituto de Salud Carlos III.

BACKGROUND Recent advances demonstrate a relationship between chronic/recurrent inflammation and prostate cancer (PCA). Among inflammatory regulators, toll-like receptors (TLRs) play a critical role in innate immune responses. However, it remains unclear whether variant TLR genes influence PCA risk among men of African descent. Therefore, we evaluated the impact of 32 TLR-associated single nucleotide polymorphisms (SNPs) on PCA risk among African-Americans and Jamaicans. METHODS SNP profiles of 814 subjects were evaluated using Illumina’s Veracode genotyping platform. Single and combined effects of SNPs in relation to PCA risk were assessed using age-adjusted logistic regression and entropy-based multifactor dimensionality reduction (MDR) models. RESULTS Seven sequence variants detected in TLR6, TOLLIP, IRAK4, IRF3 were marginally related to PCA. However, none of these effects remained significant after adjusting for multiple hypothesis testing. Nevertheless, MDR modeling revealed a complex interaction between IRAK4 rs4251545 and TLR2 rs1898830 as a significant predictor of PCA risk among U.S. men (permutation testing p-value = 0.001). CONCLUSIONS MDR identified an interaction between IRAK4 and TLR2 as the best two factor model for predicting PCA risk among men of African descent. However, these findings require further assessment and validation.

The mechanism of innate immunity is based on the pattern recognition receptors (PRR) that recognize molecular patterns associated with pathogens (PAMPs). Among PRR receptors Toll-like receptors (TLR) are distinguished. As a result of contact with pathogens, TLRs activate specific intracellular signaling pathways. It happens through proteins such as adaptor molecules, e.g. MyD88, TIRAP, TRIF, TRAM, and IPS-1, which participate in the cascade activation of kinases (IKK, MAP, RIP-1, TBK-1) as well as transcription factors (NF-κB, AP-1) and regulatory factor (IRF3). The result of this activation is the production of active proinflammatory cytokines, chemokines, interferons and enzymes. The PRR pathways are controlled by extra – and intracellular molecules to prevent overexpression of PRR. They include soluble receptors (sTLR), transmembrane proteins (ST2, SIGIRR, RP105, TRAIL-R) and intracellular inhibitors (SOCS-1, SOCS-3, sMyD88, TOLLIP, IRAK-M, SARM, A20, β-arrestin, CYLD, SHP). These molecules maintain the balance between activation and inhibition and ensure balancing of the beneficial and adverse effects of antigen recognition.

Mast cells are important cellular constituents of epithelial-mesenchymal interactions, densely located at sites of microbial entry into the host where they are continuously exposed to products from commensals. In order to avoid excessive activation and the associated pathology, mast cell responses to TLR agonists must be tightly regulated. Here, we show that exposure in vitro to subactivating levels of the epithelial cell product, IL-33, renders mast cells insensitive to bacterial cell wall products. Mast cell responsiveness to Ag, cytoplasmic dsDNA, and TLR7/8 agonists is unaffected or enhanced by IL-33. The IL-33-induced mast cell selective tolerance requires the IL-33 receptor ST2 and peritoneal mast cells from St2(-/-) mice display a constitutively activated phenotype, demonstrated by increased expression of activation markers including CD11b and CD28. IL-33 exposure neither affects the levels of TLR4, MyD88, TIRAP, IL-1R associated kinase 2 (IRAK2), or IRAK4, nor induces persistent A20 or Tollip expression, but potently causes ST2-dependent IRAK1 degradation. We show that while IRAK2 is redundant for TLR4 signaling, IRAK1 is essential for TLR4 signaling in mast cells. We suggest that IL-33 produced during homeostasis retains mast cells in an unresponsive state to bacterial cell wall products via IRAK1 degradation, thus preventing chronic inflammation and tissue destruction.

Necrotizing enterocolitis (NEC), an extensive intestinal inflammatory disease of premature infants, is caused, in part, by an excessive inflammatory response to initial bacterial colonization due to the immature expression of innate immune response genes. In a randomized placebo-controlled clinical trial, supplementation of very low birth weight infants with probiotics significantly reduced the incidence of NEC. The primary goal of this study was to determine whether secreted products of these two clinically effective probiotic strains, Bifidobacterium infantis and Lactobacillus acidophilus, prevented NEC by accelerating the maturation of intestinal innate immune response genes and whether both strains are required for this effect. After exposure to probiotic conditioned media (PCM), immature human enterocytes, immature human intestinal xenografts, and primary enterocyte cultures of NEC tissue (NEC-IEC) were assayed for an IL-8 and IL-6 response to inflammatory stimuli. The latter two models were also assayed for innate immune response gene expression. In the immature xenograft, PCM exposure significantly attenuated LPS and IL-1β-induced IL-8 and IL-6 expression, decreased TLR2 mRNA and TLR4 mRNA, and increased mRNA levels of specific negative regulators of inflammation, SIGIRR and Tollip. In NEC-IEC, PCM decreased TLR2-dependent IL-8 and IL-6 induction and increased SIGIRR and Tollip expression. The attenuated inflammatory response with PCM was reversed with Tollip siRNA-mediated knockdown. The anti-inflammatory secreted factor is a 5- to 10-kDa molecule resistant to DNase, RNase, protease, heat stress, and acid exposure. B. infantis-conditioned media showed superior anti-inflammatory properties to that of L. acidophilus in immature human enterocytes, suggesting a strain specificity to this effect. We conclude that PCM promotes maturation of innate immune response gene expression, potentially explaining the protective effects of probiotics in clinical NEC.

The endometrium is easily contaminated with bacteria and the endometrial epithelial cells (EECs) play an important role in defence against invading pathogens which recognized pathogen-associated molecular patterns (PAMPs) via pattern recognition receptors (PRRs). Toll-like receptor 4 (TLR4) can recognize lipopolysaccharide (LPS) from Gram-negative bacteria and initiates innate immune responses. In this study, we stimulated bovine EECs with LPS from Escherichia coli (E. coli). The expression of TLR4 was detected by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. The expression of downstream TLR4 signaling molecules was detected by qRT-PCR. The results showed that TLR4 and downstream adaptor molecules, transcription factors and cytokines were up-regulated when bovine EECs were stimulated with LPS. Furthermore, the expression of TOLLIP and β-defensin 5 were up-regulated when cells were stimulated with LPS. The results demonstrated that both MyD88 dependent and independent pathways in TLR4 were activated by LPS in bovine EECs. Bovine EECs have the immune repertoires required in defending against E. coli and play an important role in innate immune defence of the bovine endometrium.

Toll-like receptors (TLR) are essential for Helicobacter pylori (Hp) recognition and subsequent innate and adaptive immunity responses. TLR2 appears to be the receptor responsible for most of the immunologic reaction against Hp infection. However, TLR4, TLR9 and eventually TLR5 may also have a synergic effect with TLR2 against Hp. It has been shown that gastric Hp infection increases TLR expression in the gastric mucosa. Moreover, recent studies have shown that human gastric carcinogenesis is associated not only with increased expression of TLR but also with decreased expression of their inhibitors such as Toll-Interacting Protein (TOLLIP) and peroxisome proliferator-activated receptor (PPAR)-g. Indeed, gastric dysplasia and adenocarcinoma are associated with high expression levels of TLR and low levels of TOLLIP and PPAR-g, suggesting increased activation of these receptors throughout human gastric carcinogenesis. In this article we discuss how these novels findings could be used not only for the diagnosis and prognosis of gastric lesions associated with Hp infection but also for their treatment. Specifically, we discuss the potential use of TLR agonists in addition to antibiotics to improve eradication rates of Hp and of TLR antagonists to slow the progression of gastric preneoplastic lesions. We also discuss the potential value of TLR signalling blockers and quantification of tumoral TLR expression, respectively, in the treatment and prognosis of gastric cancer. In conclusion, TLRs can be an important link between Hp and the sequence of gastric carcinogenesis and they can be used as biomarkers of gastric carcinogenesis. In this article, future lines of investigation related with these novel scientific findings are proposed and discussed.

In addition to their classical antigen presenting functions, MHC class II molecules potentiate the TLR-triggered production of pro-inflammatory cytokines. Here, we have addressed the effect of Tollip and MARCH1 on the regulation of MHC II trafficking and TLR signaling. Our results show that MARCH1-deficient mice splenocytes are impaired in their capacity to produce pro-inflammatory cytokines in response to poly(I:C) and that TLR3 and MHC II molecules interact in the endocytic pathway. Knocking down Tollip expression in human CIITA+ HeLa cells increased expression of HLA-DR but reduced the proportion of MHC II molecules associated with the CLIP peptide. Truncation of the HLA-DR cytoplasmic tails abrogated the effect of Tollip on MHC class II expression. While overexpression of Tollip did not affect HLA-DR levels, it antagonized the function of co-transfected MARCH1. We found that Tollip strongly reduced MARCH1 protein levels and that the two molecules appear to compete for binding to MHC II molecules. Altogether, our results demonstrate that Tollip regulates MHC class II trafficking and that MARCH1 may represent a new Tollip target.