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TLR4 signaling in hepatic macrophages is increased after chronic ethanol feeding. Treatment of hepatic macrophages after chronic ethanol feeding with small-specific sized hyaluronic acid 35 (HA35) normalizes TLR4 signaling; however, the mechanisms for HA35 action are not completely understood. Here we used Next Generation Sequencing of microRNAs to identify negative regulators of TLR4 signaling reciprocally modulated by ethanol and HA35 in hepatic macrophages. Eleven microRNAs were up-regulated by ethanol; only 4 microRNAs, including miR291b, were decreased by HA35. Bioinformatics analysis identified Tollip, a negative regulator of TLR4, as a target of miR291b. Tollip expression was decreased in hepatic macrophages from ethanol-fed rats, but treatment with HA35 or transfection with a miR291b hairpin inhibitor restored Tollip expression and normalized TLR4-stimulated TNFα expression. In peripheral blood monocytes isolated from patients with alcoholic hepatitis, expression of TNFα mRNA was robustly increased in response to challenge with lipopolysaccharide. Importantly, pre-treatment with HA35 reduced TNFα expression by more than 50%. Taken together, we have identified miR291b as a critical miRNA up-regulated by ethanol. Normalization of the miR291b → Tollip pathway by HA35 ameliorated ethanol-induced sensitization of TLR4 signaling in macrophages/monocytes, suggesting that HA35 may be a novel therapeutic agent in the treatment of ALD.

Epigallocatechin-3-gallate (EGCG) was found to inhibit the Toll-like receptor 4 (TLR4) pathway involved in influenza virus pathogenesis. Here, the effect of EGCG on TLR4 in an H9N2 virus-induced acute lung injury mouse model was investigated. BALB/c mice were inoculated intranasally with A/Swine/Hebei/108/2002 (H9N2) virus or noninfectious allantoic fluid, and treated with EGCG and E5564 or normal saline orally for 5 consecutive days. PMVECs were treated with EGCG or anti-67kDa laminin receptor (LR). Lung physiopathology, inflammation, oxidative stress, viral replication, and TLR4/NF-κB/Toll-interacting protein (Tollip) pathway in lung tissue and/or PMVECs were investigated. EGCG attenuated lung histological lesions, decreased lung W/D ratio, cytokines levels, and inhibited MPO activity and prolonged mouse survival. EGCG treatment also markedly downregulated TLR4 and NF-κB protein levels but Tollip expression was upregulated compared with that in untreated H9N2-infected mice (P

Background/Aims: Epigallocatechin-3-gallate (EGCG), a major catechin found in green tea, has been shown to prevent cardiovascular diseases. Previously, Matrix metalloproteinase-9 (MMP-9), monocyte chemotactic protein-1 (MCP-1) and toll-like receptor 4 (TLR4) were confirmed to play an important role in atherosclerosis and plaque instability. Both TLR4 and its negative regulator, Toll-interacting protein (Tollip), could be mediated by EGCG. The present study aimed to examine the effect of physiological concentration of EGCG (1 µM) on the expression of MMP-9 and MCP-1 in lipopolysaccharide (LPS)-induced macrophages and the potential mechanisms underlying its actions. Methods: The RAW264.7 cell line was used. Western blot was used to determine MCP-1, TLR4, Tollip, Mitogen-activated protein kinase (MAPK) and Nuclear factor-κB (NF-κB) protein expression. MMP-9 activity was assayed by gelatine zymography. The mRNA expression of MMP-9 and MCP-1 was measured by realtime polymerase chain reaction (RT-PCR). Results: EGCG (1 µM) significantly suppressed the expression of MMP-9 and MCP-1 and inhibited MAPK and NF-κB in LPS-induced macrophages but was blocked by Tollip silencing. The expression of LPS-induced MMP-9 and MCP-1 and the phosphorylation of the ERK1/2, P38 and NF-κB pathway proteins decreased after TLR4 siRNA treatment. Furthermore, EGCG mediated TLR4 and Tollip expression through binding to 67-kDa laminin receptor (67LR). Conclusion: The results of our study suggested that EGCG (1 µM) suppresses the TLR4/MAPK/NF-κB signalling pathway, decreases the expression of the plaque instability-mediating cytokines MMP-9 and MCP-1, and might prove to be effective in stabilizing atherosclerotic plaque.

RationaleThe molecular mechanisms that regulate tuberculosis susceptibility and bacillus Calmette-Guérin (BCG)-induced immunity are mostly unknown. However, induction of the adaptive immune response is a critical step in host control of Mycobacterium tuberculosis. Toll-interacting protein (TOLLIP) is a ubiquitin-binding protein that regulates innate immune responses, including Toll-like receptor signaling, which initiate adaptive immunity. TOLLIP variation is associated with susceptibility to tuberculosis, but the mechanism by which it regulates tuberculosis immunity is poorly understood.ObjectivesTo identify functional TOLLIP variants and evaluate the role of TOLLIP variation on innate and adaptive immune responses to mycobacteria and susceptibility to tuberculosis.MethodsWe used human cellular immunology approaches to characterize the role of a functional TOLLIP variant on monocyte mRNA expression and M. tuberculosis-induced monocyte immune functions. We also examined the association of TOLLIP variation with BCG-induced T-cell responses and susceptibility to latent tuberculosis infection.Measurements and main resultsWe identified a functional TOLLIP promoter region single-nucleotide polymorphism, rs5743854, which was associated with decreased TOLLIP mRNA expression in infant monocytes. After M. tuberculosis infection, TOLLIP-deficient monocytes demonstrated increased IL-6, increased nitrite, and decreased bacterial replication. The TOLLIP-deficiency G/G genotype was associated with decreased BCG-specific IL-2+ CD4+ T-cell frequency and proliferation. This genotype was also associated with increased susceptibility to latent tuberculosis infection.ConclusionsTOLLIP deficiency is associated with decreased BCG-specific T-cell responses and increased susceptibility to tuberculosis. We hypothesize that the heightened antibacterial monocyte responses after vaccination of TOLLIP-deficient infants are responsible for decreased BCG-specific T-cell responses. Activating TOLLIP may provide a novel adjuvant strategy for BCG vaccination.

Staphylococcus aureus bloodstream infections (SABSI) are associated with high morbidity and mortality. The Toll‐like receptor 2 (TLR2) and Toll‐interacting protein (TOLLIP) are important in recognition and regulation of human innate immunity response to S. aureus. Single nucleotide polymorphisms (SNPs) in the TLR2 and TOLLIP encoding genes have been associated with disease, including BSI. The aim of this study was to examine potential associations between a selection of SNPs in the genes encoding TLR2 and TOLLIP, and predisposition, severity, and outcome of SABSI. All patients ≥18 years of age with at least one S. aureus positive blood culture collected from March 2011 through February 2014 at Akershus University Hospital, Lørenskog, Norway, were considered for inclusion. Patients attending elective orthopaedic surgery (total hip and knee replacements, lumbar surgery) served as a control group. The TLR2 Arg753Gln, TLR2 Pro631His, TOLLIP rs5743942, and rs5743867 polymorphisms were analysed using TaqMan SNP Genotyping Assays. A total of 209 SABSI patients and 295 controls were included. The TLR2 Arg753Gln and TLR2 Pro631His polymorphisms were infrequent with no homozygotes and

Background Innate immune responses induced by in vitro stimulation of primary mammary epithelial cells (MEC) using Gram-negative lipopolysaccharide (LPS) and Gram-positive lipoteichoic acid (LTA) bacterial cell wall components are well - characterized in bovine species. The objective of the current study was to characterize the downstream regulation of the inflammatory response induced by Toll-like receptors in primary goat MEC (pgMEC). We performed quantitative real-time RT-PCR (qPCR) to measure mRNA levels of 9 genes involved in transcriptional regulation or antibacterial activity: Toll-like receptor 2 (TLR2), Toll-like receptor 4 (TLR4), prostaglandin-endoperoxide synthase 2 (PTGS2), interferon induced protein with tetratricopeptide repeats 3 (IFIT3), interferon regulatory factor 3 (IRF3), myeloid differentiation primary response 88 (MYD88), nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 (NFKB1), Toll interacting protein (TOLLIP), and lactoferrin (LTF). Furthermore, we analyzed 7 cytokines involved in Toll-like receptor signaling pathways: C-C motif chemokine ligand 2 (CCL2), C-C motif chemokine ligand 5 (CCL5), C-X-C motif chemokine ligand 6 (CXCL6), interleukin 8 (CXCL8), interleukin 1 beta (IL1B), interleukin 6 (IL6), and tumor necrosis factor alpha (TNF). Results Stimulation of pgMEC with LPS for 3 h led to an increase in expression of CCL2, CXCL6, IL6, CXCL8, PTGS2, IFIT3, MYD88, NFKB1, and TLR4 (P

The changes in molecular structure and anti-inflammatory action of a gamma-irradiated quercetin were examined. Quercetin was gamma-irradiated at doses of 0, 15, 30, 50, 100 and 150 kGy, which induced new radiolytic peaks (the highest radiolytic peak at a dose of 30 kGy). Treatment of intact- and gamma-irradiated quercetin did not induce a significant cellular toxicity of macrophages at concentrations ranging from 12.5 to 50 μM. Treatment of LPS-stimulated macrophages with gamma-irradiated quercetin (30 kGy) showed a higher inhibitory action than intact-quercetin groups in the excessive expression of inducible nitric oxide synthases-mediated nitric oxide, prostaglandin E2, pro-inflammatory cytokines level, such as tumor necrosis factor-α, interleukin-6 and interleukin-1β, reactive oxygen species, as well as cell surface molecules (CD80, CD86, and MHC class I/II). The inhibition of LPS-stimulated pro-inflammatory mediators was mediated through a suppression of mitogen-activated protein kinases and nuclear factor-κB pathways. In addition, gamma-irradiated quercetin (30 kGy) markedly elevated the expression of the Toll-interacting protein compared to intact-quercetin. The inhibitory action of intact- and gamma-irradiated quercetin on the production of IL-6 and TNF-α was not observed in the down-regulation of Tollip. Therefore, these findings represent new insights into the understanding of the changes in molecular structure and the physiological properties of natural products through the application of radiation technology.

Kumazoe, M., M. Yamashita, Y. Nakamura, K. Takamatsu, J. Bae, S. Yamashita, S. Yamada, H. Onda, T. Nojiri, K. Kangawa, and H. Tachibana. 2017. Green tea polyphenol EGCG upregulates Tollip expression by suppressing Elf-1 expression. J. Immunol. 199: [3261–3269][1]. During preparation of the

The antimicrobial peptide LL-37 neutralizes the biological activity of lipopolysaccharide (LPS), while it upregulates the expression of several immune-related genes. We investigated the effect of LL-37 on gene regulation of human gingival fibroblasts (HGFs), stimulated with or without Porphyromonas gingivalis-derived LPS, a ligand for Toll-like receptor (TLR). LL-37 was non-toxic to HGFs up to a concentration of 10 μg/ml. P. gingivalis LPS upregulated the expression of IL8, CXCL10, and CCL2, whereas LL-37 reduced this upregulation. In absence of LPS, LL-37 itself upregulated the expression of IL8 and CCL2. LL-37 increased the expression of P2X7, which was constitutively expressed in HGFs. The P2X7 antagonist A-438079 suppressed the cytotoxicity and upregulatory effect of LL-37 on chemokine response, but not its downregulatory effect on P. gingivalis LPS–induced chemokine response. Whether LL-37 alters the expression of 29 genes that encode TLR-associated proteins, including TLRs, co-receptors, signaling molecules, and negative regulators, in HGFs, under stimulation with LPS, was examined. Among TLRs, P. gingivalis LPS upregulated the level of TLR4, whereas LL-37 reduced it. In co-receptors, LL-37 downregulated the level of CD14. Among signaling molecules, LL-37 augmented the LPS-upregulated expression of IRAK1. Similar effects were observed in the specific negative regulators TNFAIP3, RNF216, TOLLIP, and SIGIRR. Our results suggest that LL-37 exerts cytotoxicity and upregulation of chemokine response via the P2X7 receptor, while it induces downregulation of P. gingivalis LPS–induced chemokine response through alteration in the expression of 7 specific TLR-associated genes: downregulation of TLR4 and CD14 and upregulation of IRAK1, TNFAIP3, RNF216, TOLLIP, and SIGIRR.

Probiotic bacteria have the ability to modulate host immune responses and have potent therapeutic functional effects against several diseases, including inflammatory diseases. However, beneficial effects of probiotics are strain specific and their interactions with host immune cells to modulate inflammatory response are largely unknown. Intestinal epithelial cells (IECs), which are the first line of defense against invading pathogens, and connects between commensals/probiotics and immune system; therefore, in this study, we used human IECs to assess the probiotic effects of three selected Lactobacillus strains in vitro. An HT-29 colonic epithelial cell and HT-29/blood mononuclear cells co-culture system were stimulated with Lactobacillus followed by Salmonella for different hours, after which the mRNA level of cytokines, β-defensin-2 and negative regulators for TLR signaling and protein levels of ZO-1 and IκB-α were analyzed by real-time polymerase chain reaction and western blot analysis. L. brevis decreased Salmonella induced IL-6, IL-8, MCP-1 and IL-1β levels, whereas L. pentosus suppressed IL-6 and MCP-1 in HT-29 cells. Moreover, L. brevis was able to increase the mRNA levels of A20, Tollip, SIGIRR and IRAKM, while L. pentosus reduced the levels of A20, and IRAKM in response to Salmonella. In addition, decrease in protein level of TNF-α and increase in mRNA level of IL-10 was observed in L. brevis and L. pentosus treated HT-29 cells. Lactobacillus strains were differentially modulated ZO-1 and p-IκB-α in HT-29 cells treated with Salmonella. Overall, the results of this study indicate that Lactobacillus strains attenuate Salmonella induced inflammatory responses through beneficial modulation of TLR negative regulators and the NF-κB pathway.

Background Increasing evidence indicates that microRNAs (miRNAs) are involved in inflammatory response and immune regulation following pathogen invasion. The purpose of this study was to elucidate the roles played by Gallus gallus microRNA-1306-5p (gga-miR-1306-5p) in host responses against potential invasion by Salmonella enteritidis (SE) in chickens and the underlying mechanisms. Results In present study, the expression levels of gga-miR-1306-5p were determined in both tissues and HD11 cells. The results showed that gga-miR-1306-5p was significantly increased following SE infection or lipopolysaccharide (LPS) stimulation. The dual luciferase reporter assay further validated that gga-miR-1306-5p targeted the Toll-interacting protein (Tollip), and thereby participated in the regulation of immune response against SE or LPS stimulation through binding with the 3′-untranslated region (3’UTR) of Tollip. Additionally, the expression of Tollip was significantly blocked by over-expressed gga-miR-1306-5p. The underlying mechanisms by which gga-miR-1306-5p modulated the production of pro-inflammatory cytokines were also investigated. Molecular biological assays demonstrated that overexpression of gga-miR-1306-5p promoted the production of pro-inflammatory mediators, including NF-κB, TNF-α, IL-6, and IL-1β, which produced effects similar to those of Tollip knockdown. Conclusions Taken together, gga-miR-1306-5p induced by SE or LPS, regulates the immune response by inhibiting Tollip, which activates the production of inflammatory cytokines. This study has provided the first direct evidence that gga-miR-1306-5p targets Tollip, and is involved in the host response against SE.

Mutations in several proteins functioning as endolysosomal components cause monogenic autoimmune diseases, of which pathogenesis is linked to increased endoplasmic reticulum stress, inefficient autophagy, and defective recycling of immune receptors. We report here a heterozygous TOM1 p.G307D missense mutation, detected by whole-exome sequencing, in two related patients presenting with early-onset autoimmunity, antibody deficiency, and features of combined immunodeficiency. The index patient suffered from recurrent respiratory tract infections and oligoarthritis since early teens, and later developed persistent low-copy EBV-viremia, as well as an antibody deficiency. Her infant son developed hypogammaglobulinemia, autoimmune enteropathy, interstitial lung disease, profound growth failure, and treatment-resistant psoriasis vulgaris. Consistent with previous knowledge on TOM1 protein function, we detected impaired autophagy and enhanced susceptibility to apoptosis in patient-derived cells. In addition, we noted diminished STAT and ERK1/2 signaling in patient fibroblasts, as well as poor IFN-γ and IL-17 secretion in T cells. The mutant TOM1 failed to interact with TOLLIP, a protein required for IL-1 recycling, PAMP signaling and autophagosome maturation, further strengthening the link between the candidate mutation and patient pathophysiology. In sum, we report here an identification of a novel gene, TOM1, associating with early-onset autoimmunity, antibody deficiency, and features of combined immunodeficiency. Other patient cases from unrelated families are needed to firmly establish a causal relationship between the genotype and the phenotype.

LPS inhibits [Formula: see text] 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-β (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 [Formula: see text] 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.

Macrophages (Mϕs) play a central role in mucosal immunity by pathogen sensing and instruction of adaptive immune responses. Prior challenge to endotoxin can render Mφs refractory to secondary exposure, suppressing the inflammatory response. Previous studies demonstrated a differential subset-specific sensitivity to endotoxin tolerance (ET), mediated by LPS from the oral pathogen, Porphyromonas gingivalis (PG). The aim of this study was to investigate ET mechanisms associated with Mφ subsets responding to entropathogenic E. coli K12-LPS. M1- and M2-like Mφs were generated in vitro from the THP-1 cell line by differentiation with PMA and Vitamin D3, respectively. This study investigated ET mechanisms induced in M1 and M2 Mφ subsets, by measuring modulation of expression by RT-PCR, secretion of cytokines by sandwich ELISA, LPS receptor, TLR4, as well as endogenous TLR inhibitors, IRAK-M and Tollip by Western blotting. In contrast to PG-LPS tolerisation, E. coli K12-LPS induced ET failed to exhibit a subset-specific response with respect to the pro-inflammatory cytokine, TNFα, whereas exhibited a differential response for IL-10 and IL-6. TNFα expression and secretion was significantly suppressed in both M1- and M2-like Mφs. IL-10 and IL-6, on the other hand, were suppressed in M1s and refractory to suppression in M2s. ET suppressed TLR4 mRNA, but not TLR4 protein, yet induced differential augmentation of the negative regulatory molecules, Tollip in M1 and IRAK-M in M2 Mφs. In conclusion, E. coli K12-LPS differentially tolerises Mφ subsets at the level of anti-inflammatory cytokines, associated with a subset-specific divergence in negative regulators and independent of TLR4 down-regulation.

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.

STING (stimulator of interferon genes) is an important innate immune protein, but its homeostatic regulation at the resting state is unknown. Here, we identified TOLLIP as a stabilizer of STING through direct interaction to prevent its degradation. Tollip deficiency results in reduced STING protein in nonhematopoietic cells and tissues, and renders STING protein unstable in immune cells, leading to severely dampened STING signaling capacity. The competing degradation mechanism of resting-state STING requires IRE1α and lysosomes. TOLLIP mediates clearance of Huntington's disease-linked polyQ protein aggregates. Ectopically expressed polyQ proteins in vitro or endogenous polyQ proteins in Huntington's disease mouse striatum sequester TOLLIP away from STING, leading to reduced STING protein and dampened immune signaling. Tollip-/- also ameliorates STING-mediated autoimmune disease in Trex1-/- mice. Together, our findings reveal that resting-state STING protein level is strictly regulated by a constant tug-of-war between 'stabilizer' TOLLIP and 'degrader' IRE1α-lysosome that together maintain tissue immune homeostasis.

Hepatic ischemia-reperfusion (IR) injury is the leading cause of liver dysfunction and failure after liver resection or transplantation and lacks effective therapeutic strategies. Here, we applied a systematic proteomic analysis to identify the prominent contributors to IR-induced liver damage and promising therapeutic targets for this condition. Based on an unbiased proteomic analysis, we found that toll-interacting protein (Tollip) expression was closely correlated with the hepatic IR process. RNA sequencing analysis and phenotypic examination showed a dramatically alleviated hepatic IR injury by Tollip deficiency both in vivo and in hepatocytes. Mechanistically, Tollip interacts with apoptosis signal-regulating kinase 1 (ASK1) and facilitates the recruitment of tumor necrosis factor receptor-associated factor 6 (TRAF6) to ASK1, leading to enhanced ASK1 N-terminal dimerization and the subsequent activation of downstream mitogen-activated protein kinase (MAPK) signaling. Furthermore, the Tollip methionine and phenylalanine motif and TRAF6 ubiquitinating activity are required for Tollip-regulated ASK1-MAPK axis activation. Conclusion: Tollip is a regulator of hepatic IR injury by facilitating ASK1 N-terminal dimerization and the resultant c-Jun N-terminal kinase/p38 signaling activation. Inhibiting Tollip or its interaction with ASK1 might be promising therapeutic strategies for hepatic IR injury.

Although the importance of the tumor immune environment for the modulation of tumorigenesis and tumor regression is becoming increasingly clear, most of the research related to tumor-immune therapies has focused on adaptive immune cells, while the role and regulation of innate leukocytes such as neutrophils remains controversial and less defined. Here we observed that the selective deletion of Tollip, a key innate immune-cell modulator, led to enhanced tumor immune surveillance in a chemically induced colorectal cancer model. Tollip-deficient neutrophils significantly elevated T cell activation through enhanced expression of the costimulatory molecule CD80, and reduced expression of the inhibitory molecule PD-L1. Mechanistically, Tollip deficiency increased STAT5 and reduced STAT1, the transcription factors responsible for the expression of CD80 and PD-L1, respectively. Through adoptive transfer, we demonstrate that Tollip-deficient neutrophils, but not Tollip-deficient monocytes, are sufficient to drive enhanced tumor immune surveillance and reduced colorectal cancer burden in vivo. Our data reveal a strategy for the reprogramming of neutrophil functions conducive for the enhancement of the antitumor immune environment.

Rhinovirus is a common picornavirus with over 150 serotypes and three species, which is responsible for half of the human common cold cases. In people with chronic respiratory conditions and elders, it may also cause life-threatening diseases. Transmission routes are not definitively established but may involve direct human-to-human and indirect transmission (surfaces and aerosols based). In the present study, year-long presence of virus was tested by qPCR in the nostrils of young healthy volunteers and indoor and outdoor air samples. Results were correlated to atmospheric conditions (meteorological and air quality parameters) and voluntaries immune system-related genetic polymorphisms (TOLLIP rs5743899, IL6 rs1800795, IL1B rs16944, TNFA rs1800629) typed by PCR-RFLP. Nasal samples showed increased frequency and viral titers of Rhinovirus in spring and autumn. No indoor air samples tested positive for Rhinovirus, whereas outdoor air samples tested positive in late autumn. Sun radiation, atmospheric SO2, and benzene levels correlated with nostrils Rhinovirus detection. Both IL6 and TOLLIP polymorphisms but not TNFA or IL1B influenced Rhinovirus detection in the nostrils of voluntaries. Taken together, the results indicate that Rhinovirus circulation is determined by environmental conditions (weather, air-borne virus, and air pollution) and genetically encoded individual variation in immunity.

Idiopathic pulmonary fibrosis (IPF) is a chronic, debilitating, fibrotic lung disease leading to respiratory failure, and ultimately to death. It is characterized by marked heterogeneity regarding its clinical course. Despite significant progress in the understanding of its pathogenesis, the course of the disease and the response to treatment of an individual patient cannot be reliably predicted today. Areas covered: Non-invasive biomarkers, in particular serum biomarkers, for the (early) diagnosis, differential diagnosis, prognosis, and prediction of therapeutic response are described. The molecules are classified according to their involvement into alveolar epithelial cell injury, fibroproliferation, and matrix remodeling as well as immune regulation. Furthermore, genetic variants of TOLLIP, MUC-5B, and other genes associated with a differential response to treatment and with the development and/or the prognosis of IPF are reported. Expert commentary: The combination of multiple biomarkers may identify comprehensive biomarker signatures in IPF patients. This is a way to apply personalized medicine approach to patient affected by IPF in order to not only improve our ability to diagnose and treat disease but also offer the potential to detect disease at an earlier stage, when it is potentially easier to treat effectively.

Background: Genetic variants of TOLLIP and MUC5B have been reported to be associated with development and/or prognosis of idiopathic pulmonary fibrosis (IPF). Real-life experiences on the application of SNPs in the clinical management of IPF are lacking. This study was conducted to investigate the association of MUC5B and TOLLIP SNPs with disease course and outcome in a single-centre cohort. Patients and Methods: 50 IPF patients (M 43 and F 8, age 66±10 y) and 50 healthy controls (HC) (M 37, F 13, age42±2 y) were genotyped for SNPs within TOLLIP (rs3750920 and rs5743890) and MUC5B (rs35705950). Diagnosis of IPF was made according to the ATS/ERS guideline 2011. Genotype frequency was compared between IPF and HC subjects. Correlation of SNPs genotypes with survival, acute exacerbation or disease progression (a decline of ≥ 10% in FVC) was investigated. Results: TOLLIP SNPs genotype distribution did not differ between IPF and HC (p=0.6). MUC 5B T minor allele was more frequent in IPF subjects than in HC (67% vs 16% p Conclusion: While the minor allele T in MUC5B is associated with IPF, TOLLIP gene variants appear to correlate with disease progression, therefore being of potential utility to stratify IPF patients.

Legionella pneumophila (Lp) is a flagellated, intracellular bacterium that can cause Legionnaires’ disease (LD). Lp activates multiple innate immune receptors, and TOLLIP dampens MyD88-dependent signaling and may influence susceptibility to LD. We evaluated the effect of TOLLIP on innate immunity, pneumonia severity, and LD susceptibility in mouse lungs and human populations. To accomplish this, we evaluated the effect of TOLLIP on lung-specific Lp control and immune response and associated a common functional TOLLIP variant with Lp-induced innate immune responses and LD susceptibility in humans. After aerosol Lp infection, Tollip−/− mice demonstrated significantly fewer bacterial colony-forming unit and increased cytokine responses from BAL fluid. Tollip−/− macrophages also suppressed intracellular Lp replication in a flagellin-independent manner. The presence of a previously characterized, functionally active SNP associated with decreased TOLLIP mRNA transcript in monocytes was associated with increased TNF and IL-6 secretion after Lp stimulation of PBMC ex vivo. This genotype was separately associated with decreased LD susceptibility (309 controls, 88 cases, p = 0.008, OR 0.36, 95% CI 0.16–0.76) in a candidate gene association study. These results suggest that TOLLIP decreases lung-specific TLR responses to increase LD susceptibility in human populations. Better understanding of TOLLIP may lead to novel immunomodulatory therapies.

Epigallocatechin 3-O-gallate (EGCG), the major catechin in green tea, exerts several beneficial effects in vivo. A previous study showed that cyclic guanosine monophosphate (cGMP) as a cellular second messenger has been shown to be an important mediator of the effects of EGCG. In general, long-term animal experiments are required to evaluate the functionality of phytochemicals such as EGCG. Thus, it might be beneficial to establish a marker for short-term evaluation of functional phytochemicals. In this study, the use of the plasma cGMP level was evaluated as a surrogate marker for the functional effectiveness of EGCG. Blood samples were collected from 6-wk-old C57BL/6J mice from 1 to 8 h after oral administration of EGCG (30 or 100 mg/kg). The upregulation of plasma cGMP levels elicited by the oral EGCG administration was evaluated. Additionally, it was confirmed that EGCG induced the expressions of intracellular Toll interacting protein (Tollip), which is mainly controlled by a cGMP signal in the spleen. These data indicated that the plasma cGMP levels could be a screening marker for the effect of EGCG.

Isorhynchophylline (IRN) is an alkaloid with anti-inflammatory and anti-oxidative activities in cardiovascular and brain diseases, but its role in paraquat (PQ)-induced acute kidney injury (AKI) is yet unknown. The model of PQ-induced AKI in rats was established by intraperitoneal injection of PQ (25 mg/kg). We found that the tail vein injection of IRN (4 mg/kg) significantly increased the survival rate of PQ-intoxicated rats. IRN administration alleviated PQ-induced renal injury and renal dysfunction in rats, as evidenced by decreased apoptosis in renal cortex and reduced serum creatinine, serum BUN, and urine NGAL levels. Furthermore, IRN treatment improved the PQ-triggered oxidative stress in renal cortex by increasing the levels of anti-oxidant indicators (SOD activity, GSH/GSSG ratio, levels of Nrf-2, NQO-1, and HO-1 in renal cortex) and decreasing the levels of oxidative stress indexes (ROS and MDA levels in renal cortex). Interestingly, toll-interacting protein (Tollip), a negative regulator of interleukin 1 receptor associated kinase 1 (IRAK1) phosphorylation, was demonstrated to be increased by IRN injection in the renal cortex of PQ-intoxicated rats. In vitro experiments revealed that IRN protected renal tubular epithelial cells against PQ toxicity through suppressing oxidative stress and mitochondrial damage, and these protective effects were reversed by Tollip shRNA. Collectively, the present study demonstrated that IRN ameliorated PQ-induced AKI by attenuating oxidative stress and mitochondrial damage through upregulating Tollip, which provides new insights into the pathogenesis and treatment of PQ poisoning.

Toll-interacting protein (Tollip) is a pivotal negative regulator of inflammatory response. In the present study, the effects of Tollip overexpression on paraquat (PQ)-induced lung injury were explored through in vivo and in vitro investigations. Upon stimulation with PQ in mice, the expression of Tollip was down-regulated. Histopathological analysis revealed that the overexpression of Tollip significantly decreased inflammatory cell infiltration. Similarly, the levels of myeloperoxidase (MPO) and interleukin-1β (IL-1β) were lowered by Tollip overexpression in PQ-administrated mice. Besides, the overexpression of Tollip reduced reactive oxygen species (ROS) generation and malondialdehyde (MDA) level but enhanced superoxide dismutase (SOD) activity in PQ-treated A549 cells. Meanwhile, Tollip overexpression lowered the level of IL-1β and decreased the protein expressions of p-p65 in the cytoplasm and nuclear p65. Importantly, inhibition of NF-κB signaling pathway probably by decreasing NF-κB p65-DNA binding activity was induced by Tollip overexpression. Taken together, Tollip overexpression attenuated PQ-initiated lung injury possibly via reduction of oxidative stress and inflammation and suppression of NF-κB signaling pathway activation, which provided some novel ideas for the treatment of lung damage mediated by PQ.

PURPOSE To investigate the protective effect and mechanism of rebamipide on NSAIDs associated intestinal injury. METHODS Intestinal injury was induced in Sprague Dawley rats by intragastric administration of diclofenac with rebamipide intervention, and LPS and TAK-242 were given intraperitoneally respectively. The expression of TLR4/NF-κB and the related proteins in the intestinal mucosa were detected. 55 patients taking NSAIDs and diagnosed as NSAIDs associated small intestinal injury were recruited as NSAIDs group. Another 55 patients without NSAIDs and no obvious abnormality in the small bowel served as the control group. RESULTS The macroscopic and histological scores of the small intestinal mucosa in the rebamipide pretreatment group were significantly lower compared to the diclofenac group (p

Sex steroid hormones have been reported to induce inflammation causing dysregulation of cytokines in prostate cancer cells. However, the underlying epigenetic mechanism has not well been studied. The objective of this study was to evaluate the effect of sex steroid hormones on epigenetic DNA methylation changes in prostate cancer cells using a signature PCR methylation array panel that correspond to 96 genes with biological function in the human inflammatory and autoimmune signals in prostate cancer. Of the 96-gene panel, 32 genes showed at least 10% differentially methylation level in response to hormonal treatment when compared to untreated cells. Genes that were hypomethylated included CXCL12, CXCL5, CCL25, IL1F8, IL13RAI, STAT5A, CXCR4 and TLR5; and genes that were hypermethylated included ELA2, TOLLIP, LAG3, CD276 and MALT1. Quantitative RT-PCR analysis of select genes represented in a cytokine expression array panel showed inverse association between DNA methylation and gene expression for TOLLIP, CXCL5, CCL18 and IL5 genes and treatment of prostate cancer cells with 5'-aza-2'-deoxycytidine with or without trichostatin A induced up-regulation of TOLLIP expression. Further analysis of relative gene expression of matched prostate cancer tissues when compared to benign tissues from individual patients with prostate cancer showed increased and significant expression for CCL18 (2.6-fold; p0.001), a modest yet significant increase in IL5 expression (1.17-fold; p=0.015), and a modest increase in CXCL5 expression (1.4-fold; p=0.25). In conclusion, our studies demonstrate that sex steroid hormones can induce aberrant gene expression via differential methylation changes in prostate carcinogenesis.

SummaryBackground Rhinovirus (RV) infection in asthma induces varying degrees of airway inflammation (e.g. neutrophils), but the underlying mechanisms remain unclear. Objective The major goal was to determine the role of genetic variation [e.g. single nucleotide polymorphisms (SNPs)] of Toll-interacting protein (Tollip) in airway epithelial responses to RV in a type 2 cytokine milieu. Methods DNA from blood of asthmatic and normal subjects was genotyped for Tollip SNP rs5743899 AA, AG and GG genotypes. Human tracheobronchial epithelial (HTBE) cells from donors without lung disease were cultured to determine pro-inflammatory and antiviral responses to IL-13 and RV16. Tollip knockout and wild-type mice were challenged with house dust mite (HDM) and infected with RV1B to determine lung inflammation and antiviral response. Results Asthmatic subjects carrying the AG or GG genotype (AG/GG) compared with the AA genotype demonstrated greater airflow limitation. HTBE cells with AG/GG expressed less Tollip. Upon IL-13 and RV16 treatment, cells with AG/GG (vs. AA) produced more IL-8 and expressed less antiviral genes, which was coupled with increased NF-κB activity and decreased expression of LC3, a hallmark of the autophagic pathway. Tollip co-localized and interacted with LC3. Inhibition of autophagy decreased antiviral genes in IL-13- and RV16-treated cells. Upon HDM and RV1B, Tollip knockout (vs. wild-type) mice demonstrated higher levels of lung neutrophilic inflammation and viral load, but lower levels of antiviral gene expression. Conclusions and Clinical Relevance Our data suggest that Tollip SNP rs5743899 may predict varying airway response to RV infection in asthma.

Alzheimer's disease (AD) is an age-related neurodegenerative disorder. Its pathological hallmarks are senile plaques (SPs), which contain extracellular deposits of amyloid β (Aβ) protein fibrils and dystrophic neurites (DNs), and neurofibrillary tangles (NFTs) containing hyperphosphorylated tau. Impairment of protein-degradation systems, including the ubiquitin-proteasome and the autophagy-lysosome systems, has been proposed as one of the causes of the accumulation of these aberrant proteins in AD brains. Tom1 (target of Myb1) was originally identified by the induction of its expression by the v-Myb oncogene and is a part of two major protein-degradation systems. The present study was conducted by immunohistochemical and immunofluorescent stainings to show that Tom1 was localized in DNs, perisomatic granules (PSGs), and NFTs in AD brains. Moreover, in DNs, Tom1 colocalized with ubiquitin, lysosomal proteins, and Tom1-related proteins (Tollip and myosin VI), which act in both protein-degradation systems via Tom1. These results indicate that Tom1 plays important roles in protein-degradation systems in AD pathogenesis.

The authors previously identified plasma plasminogen activator inhibitor-1 (PAI-1) level as a quantitative lung injury biomarker in primary graft dysfunction (PGD). They hypothesized that plasma levels of PAI-1 used as a quantitative trait could facilitate discovery of genetic loci important in PGD pathogenesis. A two-stage cohort study was performed. In stage 1, they tested associations of loci with PAI-1 plasma level using linear modeling. Genotyping was performed using the Illumina CVD Bead Chip v2. Loci meeting a p < 5 × 10(-4) cutoff were carried forward and tested in stage 2 for association with PGD. Two hundred ninety-seven enrollees were evaluated in stage 1. Six loci, associated with PAI-1, were carried forward to stage 2 and evaluated in 728 patients. rs3168046 (Toll interacting protein [TOLLIP]) was significantly associated with PGD (p = 0.006). The increased risk of PGD for carrying at least one copy of this variant was 11.7% (95% confidence interval 4.9-18.5%). The false-positive rate for individuals with this genotype who did not have PGD was 6.1%. Variants in the TOLLIP gene are associated with higher circulating PAI-1 plasma levels and validate for association with clinical PGD. A protein quantitative trait analysis for PGD risk prioritizes genetic variations in TOLLIP and supports a role for Toll-like receptors in PGD pathogenesis.

ERBB2 overexpression in human breast cancer leads to invasive carcinoma but the mechanism is not clearly understood. Here we report that TOM1L1 is co-amplified with ERBB2 and defines a subgroup of HER2+/ER+ tumours with early metastatic relapse. TOM1L1 encodes a GAT domain-containing trafficking protein and is a SRC substrate that negatively regulates tyrosine kinase signalling. We demonstrate that TOM1L1 upregulation enhances the invasiveness of ERBB2-transformed cells. This pro-tumoural function does not involve SRC, but implicates membrane-bound membrane-type 1 MMP (MT1-MMP)-dependent activation of invadopodia, membrane protrusions specialized in extracellular matrix degradation. Mechanistically, ERBB2 elicits the indirect phosphorylation of TOM1L1 on Ser321. The phosphorylation event promotes GAT-dependent association of TOM1L1 with the sorting protein TOLLIP and trafficking of the metalloprotease MT1-MMP from endocytic compartments to invadopodia for tumour cell invasion. Collectively, these results show that TOM1L1 is an important element of an ERBB2-driven proteolytic invasive programme and that TOM1L1 amplification potentially enhances the metastatic progression of ERBB2-positive breast cancers., ERBB2 overexpression in human breast cancer leads to invasion and metastasis. Here the authors report that ERBB2 induces indirect phosphorylation of TOM1L1 that promotes trafficking of the metalloprotease MT1-MMP to invadopodia, which leads to tumour cell invasion.

Cellular homeostasis requires correct delivery of cell-surface receptor proteins (cargo) to their target subcellular compartments. The adapter proteins Tom1 and Tollip are involved in sorting of ubiquitinated cargo in endosomal compartments. Recruitment of Tom1 to the endosomal compartments is mediated by its GAT domain’s association to Tollip’s Tom1-binding domain (TBD). In this data article, we report the solution NMR-derived structure of the Tom1 GAT domain. The estimated protein structure exhibits a bundle of three helical elements. We compare the Tom1 GAT structure with those structures corresponding to the Tollip TBD- and ubiquitin-bound states. Keywords: Tom1, GAT domain, Tollip, Ubiquitin, nuclear magnetic resonance

Aetiology of the establishment of Leishmania donovani infection in its host is still not well understood. Host immune system plays a key role during pathogen entry, survival and propagation of the parasite. Toll-like receptors and its signalling pathways are the hallmarks of the innate immune system and Toll interacting protein (Tollip) is a key regulatory protein of this pathway. Genetic polymorphisms of Tollip gene are known to be associated with a wide range of bacterial and protozoan diseases. However, its role in Visceral Leishmaniasis (VL) as a risk factor is not known. Therefore, we carried out a case-control study to evaluate the role of Tollip gene polymorphism with VL in an endemic population of Assam, India. A total of 206 participants (70 cases and 136 healthy controls) were enrolled in this study. The two Tollip gene polymorphisms rs3750920 and rs5743899 were genotyped by the PCR-RFLP method. The findings show no evidence of deviation from Hardy-Weinberg equilibrium in either of the kala-azar cases or healthy controls. In Tollip variants the rs3750920 &rs5743899 genotype frequency was found within acceptable HWE limits (p-value >0.05). Genetic association of the genotypes of rs3750920 did not show any association with VL. In case of rs5743899, the risk of VL due to allele G was found to be 1.859 times (p-value 2-fold with an OR of 2.228 (p-value

Summary Expression of Toll-interacting protein (Tollip), a potent TLR modulator, decreases in patients with inflammatory bowel diseases (IBD), whereas Tollip−/− mice are susceptible to colitis. Tollip expression was shown to be reduced in sporadic adenoma . In contrast, we found variable Tollip expression in patients with colitis-associated adenomas. In Tollip−/− mice challenged to develop colitis-associated cancer (CAC), tumor formation was significantly reduced owing to decreased mucosal proliferative and apoptotic indexes. This protection was associated with blunt inflammatory responses without significant changes in microbial composition. mRNA expression of Cd62l and Ccr5 homing receptors was reduced in colons of untreated Tollip−/− mice, whereas CD62L+ CD8+ T cells accumulated in the periphery. In Tollip-deficient adenomas Ctla-4 mRNA expression and tumor-infiltrating CD4+ Foxp3+ regulatory T cell (Treg) were decreased. Our data show that protection from CAC in Tollip-deficient mice is associated with defects in lymphocyte accumulation and composition in colitis-associated adenomas., Graphical Abstract, Highlights • Tollip protects from colitis but promotes colitis-associated cancer onset • Tollip-deficient tumors demonstrate decreased cell turnover and inflammation • Tollip ablation favors naive CD8+ T cell accumulation in peripheral lymphoid organs • Regulatory T cell accumulation is aberrant in Tollip-deficient tumors, Biological Sciences; Immunology; Cancer

Although the novel resveratrol derivative RM has therapeutic potential for the treatment of inflammatory bowel disease, little is currently known regarding the manner whereby RM regulates excessive inflammatory responses. In this study, we initially investigated the molecular mechanisms underlying the anti-inflammatory effects induced by RM in Toll-like receptor (TLR)-activated macrophages. Upon stimulation with lipopolysaccharide, we found that RM-treated activated macrophages down-regulated the increase in pro-inflammatory cytokines (TNF-α, IL-6, IL-1β, and IL-12p70), nitric oxide (NO) production, and activating interleukin-1 receptor-associated kinase 1 (IRAK-1) phosphorylation, mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) signaling pathways. Interestingly, the TLR negative regulator Toll-interacting protein (Tollip) was selectively enhanced during RM stimulation in time- and dose-dependent manners. In response to knockdown of Tollip expression by RNA interference, RM-treated activated macrophages showed augmented expression of inflammatory mediators (pro-inflammatory cytokines, NO, inducible nitric oxidase, and cyclooxygenase-2, and surface molecules) and restored the expression of MAPK and NF-κB signals inhibited by RM treatment. Taken together, our findings indicate that RM has therapeutic potential for treating TLR-induced inflammatory diseases via the promotion of Tollip expression.

Parasite recognition by Toll-like receptors (TLRs) contributes to macrophage activation and subsequent control of Leishmania infection through the coordinated production of pro-inflammatory and microbicidal effector molecules. The modulation of microRNA (miRNA) expression by Leishmania infection potentially mediates the post-transcriptional regulation of the expression of genes involved in leishmanicidal activity. Here, the contribution of TLR signaling to the miRNA profile and gene expression was evaluated in Leishmania amazonensis-infected murine macrophages. The infectivity of L. amazonensis was higher in murine bone marrow-derived macrophages from mice knockout for myeloid differentiation factor 88 (MyD88−/−), TLR2 (TLR2−/−), or TLR4 (TLR4−/−) than wild type C57BL/6 (WT). L. amazonensis infection of WT macrophages modulated the expression of 32% of the miRNAs analyzed, while 50% were upregulated. The absence of MyD88, TLR2, and TLR4 altered the percentage of miRNAs modulated during L. amazonensis infection, including the downregulation of let-7e expression. Moreover, the absence of signals mediated by MyD88, TLR2, or TLR4 reduced nitric oxide synthase 2 (Nos2) mRNA expression during infection. Indeed, the inhibition of let-7e increased levels of the Nos2 mRNA and NOS2 (or iNOS) protein and nitric oxide (NO) production in L. amazonensis-infected macrophages (4–24 h). The absence of TLR2 and inhibition of let-7e increased the expression of the arginase 1 (Arg1) mRNA but did not alter the protein level during infection. However, higher levels of the L-arginine transporters Cat2B and Cat1 were detected in the absence of Myd88 signaling during infection but were not altered following let-7e inhibition. The inhibition of let-7e impacted the global expression of genes in the TLR pathway by upregulating the expression of recognition and adaptors molecules, such as Tlr6, Tlr9, Ly96, Tirap, Traf 6, Ticam1, Tollip, Casp8, Map3k1, Mapk8, Nfkbib, Nfkbil1, Ppara, Mapk8ip3, Hspd1, and Ube2n, as well as immunomodulators, such as Ptgs2/Cox2, Csf 2, Csf 3, Ifnb1, Il6ra, and Ilr1, impacting NOS2 expression, NO production and parasite infectiveness. In conclusion, L. amazonensis infection alters the TLR signaling pathways by modulating the expression of miRNAs in macrophages to subvert the host immune responses.

[Purpose] Despite numerous studies, the mechanisms underlying the effects of exercise on brain function are not yet fully understood. Adult hippocampal neurogenesis is one of the most well-known effects of exercise on the brain, but its physiological roles during exercise are still ambiguous, mostly due to the difference in the structure and composition of each part of the hippocampus. [Methods] In this study, we analyzed exercise-induced changes in gene expression in the subgranular zone (SGZ) and granular cell layer (GCL) of the hippocampus. [Results] Surprisingly, only about 10% of changes were common to both areas. Tollip expression, which is altered in the SGZ and in Engrailed-2 mutant mice following exercise, did not change in the GCL. Tollip levels were not changed in the whole hippocampus after two weeks of treadmill exercise, but immunofluorescence analysis showed that Tollip and Ki-67 co-localize in the hippocampal dentate gyrus . Through siRNA knockdown experiments, we found that levels of DCX and cellular survival rates were decreased in Tollip-deficient Neuro2A cells. [Conclusion] Taken together, these results suggest a role for Tollip in mediating the beneficial effects of exercise, probably affecting cellular health in the SGZ of the hippocampus.

Lysosomes are highly dynamic organelles that can move rapidly throughout the cell. They distribute in a rather immobile pool located around the microtubule‐organizing center in a “cloud,” and a highly dynamic pool in the cell periphery. Their spatiotemporal characteristics allow them to carry out multiple biological functions, such as cargo degradation, antigen presentation and plasma membrane repair. Therefore, it is not surprising that lysosomal dysfunction underlies various diseases, including cancer, neurodegenerative and autoimmune diseases. In most of these biological events, the involvement of lysosomes is dependent on their ability to move throughout the cytoplasm, to find and fuse to the correct compartments to receive and deliver substrates for further handling. These dynamics are orchestrated by motor proteins moving along cytoskeletal components. The complexity of the mechanisms responsible for controlling lysosomal transport has recently been appreciated and has yielded novel insights into interorganellar communication, as well as lipid‐protein interplay. In this review, we discuss the current understanding of the mechanisms of lysosomal transport and the molecular machineries that control this mobility.

Inflammatory bowel diseases (IBD), encompassing both Crohn Disease (CD) and ulcerative colitis (UC) are globally prevalent diseases, impacting children of all ages. The hallmark of IBD is a perturbed immune system that leads to continuous inflammation in the gut and challenges optimal treatment. Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κβ), a nuclear transcription factor, plays a major role in gut homeostasis and contributes significantly toward a balanced, homeostatic immune system. Dysregulation in the NF-κβ pathway and factors that regulate it lead to a state of uncontrolled inflammation and altered immunity, as typically observed in IBD. Levels of proinflammatory cytokines that are regulated through NF-κβ are increased in both CD and UC. Genes known to activate NF-κβ, such as, Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) and Interleukin 23 (IL-23), are associated with IBD. Factors involved in inhibition of NF-κβ, such as A20 and TOLLIP, are also affected in IBD, resulting in failed inflammation suppression/regulation. NOD-2 and A20 have specifically been found to be strongly associated with pediatric IBD. Gut commensals are known to exert anti-inflammatory activities toward NF-κβ and can have a potential role in attenuating inflammation that likely occurs due to microbial dysbiosis in IBD. Failure to terminate/downregulate NF-κβ signaling results in chronic inflammation in IBD. Well-regulated control of inflammation in children with IBD can help better control the disease and suppress immune responses. Better understanding of factors that control NF-κβ can potentially lead toward discovering targeted therapeutic interventions for IBD. Suppression of NF-κβ can be achieved through many modalities including anti-sense oligonucleotides (ASOs), siRNA (small interfering RNA), factors regulating NF-κβ, and microbes. This review focuses on the role of NF-κβ, especially in pediatric IBD, and potential therapeutic venues for attenuating NF-κβ-induced inflammation.

Raf kinase inhibitor protein (RKIP) plays a critical role in many signaling pathways as a multi-functional adapter protein. In particular, the loss of RKIP’s function in certain types of cancer cells results in epithelial to mesenchymal transition (EMT) and the promotion of cancer metastasis. In addition, RKIP inhibits autophagy by modulating LC3-lipidation and mTORC1. How the RKIP-dependent inhibition of autophagy is linked to EMT and cancer progression is still under investigation. In this study, we investigated the ways by which RKIP interacts with key gene products in EMT and autophagy during the progression of prostate cancer. We first identified the gene products of interest using the corresponding gene ontology terms. The weighted-gene co-expression network analysis (WGCNA) was applied on a gene expression dataset from three groups of prostate tissues, benign prostate hyperplasia, primary and metastatic cancer. We found two modules of highly co-expressed genes, which were preserved in other independent datasets of prostate cancer tissues. RKIP showed potentially novel interactions with one EMT and seven autophagy gene products (TGFBR1, PIK3C3, PIK3CB, TBC1D25, TBC1D5, TOLLIP, WDR45 and WIPI1). In addition, we identified several upstream transcription modulators that could regulate the expression of these gene products. Finally, we verified some RKIP novel interactions by co-localization using the confocal microscopy analysis in a prostate cancer cell line. To summarize, RKIP interacts with EMT and autophagy as part of the same functional unit in developing prostate cancer.