Cytochrome P450 1A1 is essential for the microbial metabolite, Urolithin A-mediated protection against colitis.

Background: Cytochrome P450 Family 1 Subfamily A Member 1 (CYP1A1) pathway, which is regulated by aryl hydrocarbon receptor (AhR) plays an important role in chemical carcinogenesis and xenobiotic metabolism. Recently, we demonstrated that the microbial metabolite Urolithin A (UroA) mitigates colitis through its gut barrier protective and anti-inflammatory activities in an AhR-dependent manner. Here, we explored role of CYP1A1 in UroA-mediated gut barrier and immune functions in regulation of inflammatory bowel disease (IBD). Methods: To determine the role of CYP1A1 in UroA-mediated protectives activities against colitis, we subjected C57BL/6 mice and Cyp1a1 -/- mice to dextran sodium sulphate (DSS)-induced acute colitis model. The phenotypes of the mice were characterized by determining loss of body weight, intestinal permeability, systemic and colonic inflammation. Further, we evaluated the impact of UroA on regulation of immune cell populations by flow cytometry and confocal imaging using both in vivo and ex vivo model systems. Results: UroA treatment mitigated DSS-induced acute colitis in the wildtype mice. However, UroA-failed to protect Cyp1a1 -/- mice against colitis, as evident from non-recovery of body weight loss, shortened colon lengths and colon weight/length ratios. Further, UroA failed to reduce DSS-induced inflammation, intestinal permeability and upregulate tight junction proteins in Cyp1a1 -/- mice. Interestingly, UroA induced the expansion of T-reg cells in a CYP1A1-dependent manner both in vivo and ex vivo models. Conclusion: Our results suggest that CYP1A1 expression is essential for UroA-mediated enhanced gut barrier functions and protective activities against colitis. We postulate that CYP1A1 plays critical and yet unknown functions beyond xenobiotic metabolism in the regulation of gut epithelial integrity and immune systems to maintain gut homeostasis in IBD pathogenesis.

Microbial metabolite restricts 5-fluorouracil-resistant colonic tumor progression by sensitizing drug transporters via regulation of FOXO3-FOXM1 axis.

The survival rate of colorectal cancer patients is adversely affected by the selection of tumors resistant to conventional anti-cancer drugs such as 5-fluorouracil (5FU). Although there is mounting evidence that commensal gut microbiota is essential for effective colon cancer treatment, the detailed molecular mechanisms and the role of gut microbial metabolites remain elusive. The goal of this study is to decipher the impact and mechanisms of gut microbial metabolite, urolithin A (UroA) and its structural analogue, UAS03 on reversal of 5FU-resistant (5FUR) colon cancers. Methods: We have utilized the SW480 and HCT-116 parental (5FU-sensitive) and 5FUR colon cancer cells to examine the chemosensitization effects of UroA or UAS03 by using both in vitro and in vivo models. The effects of mono (UroA/UAS03/5FU) and combinatorial therapy (UroA/UAS03 + 5FU) on cell proliferation, apoptosis, cell migration and invasion, regulation of epithelial mesenchymal transition (EMT) mediators, expression and activities of drug transporters, and their regulatory transcription factors were examined using molecular, cellular, immunological and flowcytometric methods. Further, the anti-tumor effects of mono/combination therapy (UroA or UAS03 or 5FU or UroA/UAS03 + 5FU) were examined using pre-clinical models of 5FUR-tumor xenografts in NRGS mice and azoxymethane (AOM)-dextran sodium sulfate (DSS)-induced colon tumors. Results: Our data showed that UroA or UAS03 in combination with 5FU significantly inhibited cell viability, proliferation, invasiveness as well as induced apoptosis of the 5FUR colon cancer cells compared to mono treatments. Mechanistically, UroA or UAS03 chemosensitized the 5FUR cancer cells by downregulating the expression and activities of drug transporters (MDR1, BCRP, MRP2 and MRP7) leading to a decrease in the efflux of 5FU. Further, our data suggested the UroA or UAS03 chemosensitized 5FUR cancer cells to 5FU treatment through regulating FOXO3-FOXM1 axis. Oral treatment with UroA or UAS03 in combination with low dose i.p. 5FU significantly reduced the growth of 5FUR-tumor xenografts in NRGS mice. Further, combination therapy significantly abrogated colonic tumors in AOM-DSS-induced colon tumors in mice. Conclusions: In summary, gut microbial metabolite UroA and its structural analogue UAS03 chemosensitized the 5FUR colon cancers for effective 5FU chemotherapy. This study provided the novel characteristics of gut microbial metabolites to have significant translational implications in drug-resistant cancer therapeutics.

Epidermal Fatty Acid‒Binding Protein Mediates Depilatory-Induced Acute Skin Inflammation.

Depilatory creams are widely used to remove unwanted body hair, but people with sensitive skin are subject to depilatory-induced skin burn/inflammation. It remains unknown what makes their skin more sensitive than others. In this study, we show that epidermal fatty acid‒binding protein (E-FABP) expressed in the skin plays a critical role in promoting depilatory-induced acute skin inflammation in mouse models. Although a depilatory cream removed hair by breaking down keratin disulfide bonds, it activated cytosolic phospholipase A2, leading to activation of the arachidonic acid/E-FABP/peroxisome proliferator-activated receptor ß signaling pathway in keratinocytes. Specifically, peroxisome proliferator-activated receptor ß activation induced downstream targets (e.g., cyclooxygenase 2) and chemokine (e.g., CXCL1) production, which systemically mobilized neutrophils and recruited them to localize in the skin for acute inflammatory responses. Importantly, E-FABP deletion by CRISPR-Cas9 reduced cytosolic phospholipase A2/peroxisome proliferator-activated receptor ß activation in keratinocytes, and genetic deletion of E-FABP protected mice from depilatory cream-induced neutrophil recruitment and skin inflammation. Our findings suggest E-FABP as a molecular sensor for sensitive skin by triggering depilatory-induced, lipid-mediated skin inflammatory responses.

Absence of CCR2 reduces spontaneous intestinal tumorigenesis in the ApcMin /+ mouse model.

The biological activities of chemokine (C-C motif) ligand 2 (CCL2) are mediated via C-C chemokine receptor-2 (CCR2). Increased CCL2 level is associated with metastasis of many cancers. In our study, we investigated the role of the CCL2/CCR2 axis in the development of spontaneous intestinal tumorigenesis using the ApcMin/+ mouse model. Ablation of CCR2 in ApcMin/+ mice significantly increased the overall survival and reduced intestinal tumor burden. Immune cell analysis showed that CCR2-/- ApcMin/+ mice exhibited significant reduction in the myeloid cell population and increased interferon γ (IFN-γ) producing T cells both in spleen and mesenteric lymph nodes compared to ApcMin/+ mice. The CCR2-/- ApcMin/+ tumors showed significantly reduced levels of interleukin (IL)-17 and IL-23 and increased IFN-γ and Granzyme B compared to ApcMin/+ tumors. Transfer of CCR2+/+ ApcMin/+ CD4+ T cells into Rag2-/- mice led to development of colitis phenotype with increased CD4+ T cells hyper proliferation and IL-17 production. In contrast, adoptive transfer of CCR2-/- ApcMin/+ CD4+ T cells into Rag2-/- mice failed to enhance colonic inflammation or IL-17 production. These results a suggest novel additional role for CCR2, where it regulates migration of IL-17 producing cells mediating tumor-promoting inflammation in addition to its role in migration of tumor associated macrophages.

Microbial Metabolite Urolithin B Inhibits Recombinant Human Monoamine Oxidase A Enzyme.

Urolithins are gut microbial metabolites derived from ellagitannins (ET) and ellagic acid (EA), and shown to exhibit anticancer, anti-inflammatory, anti-microbial, anti-glycative and anti-oxidant activities. Similarly, the parent molecules, ET and EA are reported for their neuroprotection and antidepressant activities. Due to the poor bioavailability of ET and EA, the in vivo functional activities cannot be attributed exclusively to these compounds. Elevated monoamine oxidase (MAO) activities are responsible for the inactivation of monoamine neurotransmitters in neurological disorders, such as depression and Parkinson's disease. In this study, we examined the inhibitory effects of urolithins (A, B and C) and EA on MAO activity using recombinant human MAO-A and MAO-B enzymes. Urolithin B was found to be a better MAO-A enzyme inhibitor among the tested urolithins and EA with an IC50 value of 0.88 µM, and displaying a mixed mode of inhibition. However, all tested compounds exhibited higher IC50 (>100 µM) for MAO-B enzyme.

Zinc Oxide Nanowires Exposure Induces a Distinct Inflammatory Response via CCL11-Mediated Eosinophil Recruitment.

High aspect ratio zinc oxide nanowires (ZnONWs) have become one of the most important products in nanotechnology. The wide range applications of ZnONWs have heightened the need for evaluating the risks and biological consequences to these particles. In this study, we investigated inflammatory pathways activated by ZnONWs in cultured cells as well as the consequences of systemic exposure in mouse models. Confocal microscopy showed rapid phagocytic uptake of FITC-ZnONWs by macrophages. Exposure of macrophages or lung epithelial cells to ZnONWs induced the production of CCL2 and CCL11. Moreover, ZnONWs exposure induced both IL-6 and TNF-α production only in macrophages but not in LKR13 cells. Intratracheal instillation of ZnONWs in C57BL/6 mice induced a significant increase in the total numbers of immune cells in the broncho alveolar lavage fluid (BALFs) 2 days after instillation. Macrophages and eosinophils were the predominant cellular infiltrates of ZnONWs exposed mouse lungs. Similar cellular infiltrates were also observed in a mouse air-pouch model. Pro-inflammatory cytokines IL-6 and TNF-α as well as chemokines CCL11, and CCL2 were increased both in BALFs and air-pouch lavage fluids. These results suggest that exposure to ZnONWs may induce distinct inflammatory responses through phagocytic uptake and formation of soluble Zn2+ ions.

Co-localization of autophagy-related protein p62 with cancer stem cell marker dclk1 may hamper dclk1's elimination during colon cancer development and progression.

Autophagy may play a critical role in colon cancer stem cells (CCSCs)-related cancer development. Here, we investigate whether accumulation of infection/injury-induced CCSCs due to impaired autophagy influences colon cancer development and progression. When Apc++ mice were infected with Citrobacter rodentium (CR; 109CFUs), we discovered presence of autophagosomes with increases in Beclin-1, LC3B and p62 staining during crypt hyperplasia. Apc1638N/+ mice when infected with CR or subjected to CR+AOM treatment, exhibited increased colon tumorigenesis with elevated levels of Ki-67, ß-catenin, EZH2 and CCSC marker Dclk1, respectively. AOM/DSS treatment of Apc1638N/+ mice phenocopied CR+AOM treatment as colonic tumors exhibited pronounced changes in Ki-67, EZH2 and Dclk1 accompanied by infiltration of F4/80+ macrophages, CD3+ lymphocytes and CD3/ß-catenin co-localization. Intestinal and colonic tumors also stained positive for migrating CSC markers CD110 and CDCP1 wherein, colonic tumors additionally exhibited stromal positivity. In tumors from CR-infected, CR+AOM or AOM/DSS-treated Apc1638N/+ mice and surgically-resected colon tumor/metastatic liver samples, significant accumulation of p62 and it's co-localization with LC3B and Dclk1 was evident. ApcMin/+ mice when infected with CR and BLT1-/-;ApcMin/+ mice, exhibited similar co-localization of p62 with LC3B and Dclk1 within the tumors. Studies in HCT116 and SW480 cells further confirmed p62/Dclk1 co-localization and Chloroquin/LPS-induced increases in Dclk1 promoter activity. Thus, co-localization of p62 with Dclk1 may hamper Dclk1's elimination to impact colon cancer development and progression.

Enhancement of the gut barrier integrity by a microbial metabolite through the Nrf2 pathway.

The importance of gut microbiota in human health and pathophysiology is undisputable. Despite the abundance of metagenomics data, the functional dynamics of gut microbiota in human health and disease remain elusive. Urolithin A (UroA), a major microbial metabolite derived from polyphenolics of berries and pomegranate fruits displays anti-inflammatory, anti-oxidative, and anti-ageing activities. Here, we show that UroA and its potent synthetic analogue (UAS03) significantly enhance gut barrier function and inhibit unwarranted inflammation. We demonstrate that UroA and UAS03 exert their barrier functions through activation of aryl hydrocarbon receptor (AhR)- nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent pathways to upregulate epithelial tight junction proteins. Importantly, treatment with these compounds attenuated colitis in pre-clinical models by remedying barrier dysfunction in addition to anti-inflammatory activities. Cumulatively, the results highlight how microbial metabolites provide two-pronged beneficial activities at gut epithelium by enhancing barrier functions and reducing inflammation to protect from colonic diseases.

Inflammasome-Independent Leukotriene B4 Production Drives Crystalline Silica-Induced Sterile Inflammation.

Silicosis is a lung inflammatory disease caused by chronic exposure to crystalline silica (CS). Leukotriene B4 (LTB4) plays an important role in neutrophilic inflammation, which drives silicosis and promotes lung cancer. In this study, we examined the mechanisms involved in CS-induced inflammatory pathways. Phagocytosis of CS particles is essential for the production of LTB4 and IL-1ß in mouse macrophages, mast cells, and neutrophils. Phagosomes enclosing CS particles trigger the assembly of lipidosome in the cytoplasm, which is likely the primary source of CS-induced LTB4 production. Activation of the JNK pathway is essential for both CS-induced LTB4 and IL-1ß production. Studies with bafilomycin-A1- and NLRP3-deficient mice revealed that LTB4 synthesis in the lipidosome is independent of inflammasome activation. Small interfering RNA knockdown and confocal microscopy studies showed that GTPases Rab5c, Rab40c along with JNK1 are essential for lipidosome formation and LTB4 production. BI-78D3, a JNK inhibitor, abrogated CS-induced neutrophilic inflammation in vivo in an air pouch model. These results highlight an inflammasome-independent and JNK activation-dependent lipidosome pathway as a regulator of LTB4 synthesis and CS-induced sterile inflammation.

Mast Cell-Dependent CD8+ T-cell Recruitment Mediates Immune Surveillance of Intestinal Tumors in ApcMin/+ Mice.

The presence of mast cells in some human colorectal cancers is a positive prognostic factor, but the basis for this association is incompletely understood. Here, we found that mice with a heterozygous mutation in the adenomatous polyposis coli gene (ApcMin/+) displayed reduced intestinal tumor burdens and increased survival in a chemokine decoy receptor, ACKR2-null background, which led to discovery of a critical role for mast cells in tumor defense. ACKR2-/-ApcMin/+ tumors showed increased infiltration of mast cells, their survival advantage was lost in mast cell-deficient ACKR2-/-SA-/-ApcMin/+ mice as the tumors grew rapidly, and adoptive transfer of mast cells restored control of tumor growth. Mast cells from ACKR2-/- mice showed elevated CCR2 and CCR5 expression and were also efficient in antigen presentation and activation of CD8+ T cells. Mast cell-derived leukotriene B4 (LTB4) was found to be required for CD8+ T lymphocyte recruitment, as mice lacking the LTB4 receptor (ACKR2-/-BLT1-/-ApcMin/+) were highly susceptible to intestinal tumor-induced mortality. Taken together, these data demonstrate that chemokine-mediated recruitment of mast cells is essential for initiating LTB4/BLT1-regulated CD8+ T-cell homing and generation of effective antitumor immunity against intestinal tumors. We speculate that the pathway reported here underlies the positive prognostic significance of mast cells in selected human tumors. Cancer Immunol Res; 6(3); 332-47. ©2018 AACR.

Leukotriene B4-receptor-1 mediated host response shapes gut microbiota and controls colon tumor progression.

Inflammation and infection are key promoters of colon cancer but the molecular interplay between these events is largely unknown. Mice deficient in leukotriene B4 receptor1 (BLT1) are protected in inflammatory disease models of arthritis, asthma and atherosclerosis. In this study, we show that BLT1-/- mice when bred onto a spontaneous tumor (ApcMin/+) model displayed an increase in the rate of intestinal tumor development and mortality. A paradoxical increase in inflammation in the tumors from the BLT1-/-ApcMin/+ mice is coincidental with defective host response to infection. Germ-free BLT1-/-ApcMin/+ mice are free from colon tumors that reappeared upon fecal transplantation. Analysis of microbiota showed defective host response in BLT1-/- ApcMin/+ mice reshapes the gut microbiota to promote colon tumor development. The BLT1-/-MyD88-/- double deficient mice are susceptible to lethal neonatal infections. Broad-spectrum antibiotic treatment eliminated neonatal lethality in BLT1-/-MyD88-/- mice and the BLT1-/-MyD88-/-ApcMin+ mice are protected from colon tumor development. These results identify a novel interplay between the Toll-like receptor mediated microbial sensing mechanisms and BLT1-mediated host response in the control of colon tumor development.

The yin and yang of leukotriene B4 mediated inflammation in cancer.

The high affinity leukotriene B4 receptor, BLT1 mediates chemotaxis of diverse leukocyte subsets to the sites of infection or inflammation. Whereas the pathological functions of LTB4/BLT1 axis in allergy, autoimmunity and cardiovascular disorders are well established; its role in cancer is only beginning to emerge. In this review, we summarize recent findings on LTB4/BLT1 axis enabling distinct outcomes toward tumor progression. In a mouse lung tumor model promoted by silicosis-induced inflammation, genetic deletion of BLT1 attenuated neutrophilic inflammation and tumor promotion. In contrast, in a spontaneous model of intestinal tumorigenesis, absence of BLT1 led to defective mucosal host response, altered microbiota and bacteria dependent colon tumor progression. Furthermore, BLT1 mediated CD8+ T cell recruitment was shown to be essential for initiating anti-tumor immunity in number of xenograft models and is critical for effective PD1 based immunotherapy. BLT2 mediated chemotherapy resistance, tumor promotion and metastasis are also discussed. This new information points to a paradigm shift in our understanding of the LTB4 pathways in cancer.

Microbiota-activated CD103+ DCs stemming from microbiota adaptation specifically drive γδT17 proliferation and activation.

BACKGROUND: IL-17-producing γδT cells (γδT17) promote autoinflammatory diseases and cancers. Yet, γδT17 peripheral regulation has not been thoroughly explored especially in the context of microbiota-host interaction. The potent antigen-presenting CD103+ dendritic cell (DC) is a key immune player in close contact with both γδT17 cells and microbiota. This study presents a novel cellular network among microbiota, CD103+ DCs, and γδT17 cells. METHODS: Immunophenotyping of IL-17r-/- mice and IL-17r-/- IRF8-/- mice were performed by ex vivo immunostaining and flow cytometric analysis. We observed striking microbiome differences in the oral cavity and gut of IL-17r-/- mice by sequencing 16S rRNA gene (v1-v3 region) and analyzed using QIIME 1.9.0 software platform. Principal coordinate analysis of unweighted UniFrac distance matrix showed differential clustering for WT and IL-17r-/- mice. RESULTS: We found drastic homeostatic expansion of γδT17 in all major tissues, most prominently in cervical lymph nodes (cLNs) with monoclonal expansion of Vγ6 γδT17 in IL-17r-/- mice. Ki-67 staining and in vitro CFSE assays showed cellular proliferation due to cell-to-cell contact stimulation with microbiota-activated CD103+ DCs. A newly developed double knockout mice model for IL-17r and CD103+ DCs (IL-17r-/-IRF8-/-) showed a specific reduction in Vγ6 γδT17. Vγ6 γδT17 expansion is inhibited in germ-free mice and antibiotic-treated specific pathogen-free (SPF) mice. Microbiota transfer using cohousing of IL-17r-/- mice with wildtype mice induces γδT17 expansion in the wildtype mice with increased activated CD103+ DCs in cLNs. However, microbiota transfer using fecal transplant through oral gavage to bypass the oral cavity showed no difference in colon or systemic γδT17 expansion. CONCLUSIONS: These findings reveal for the first time that γδT17 cells are regulated by microbiota dysbiosis through cell-to-cell contact with activated CD103+ DCs leading to drastic systemic, monoclonal expansion. Microbiota dysbiosis, as indicated by drastic bacterial population changes at the phylum and genus levels especially in the oral cavity, was discovered in mice lacking IL-17r. This network could be very important in regulating both microbiota and immune players. This critical regulatory pathway for γδT17 could play a major role in IL-17-driven inflammatory diseases and needs further investigation to determine specific targets for future therapeutic intervention.

Chemoattractant Receptors BLT1 and CXCR3 Regulate Antitumor Immunity by Facilitating CD8+ T Cell Migration into Tumors.

Immunotherapies have shown considerable efficacy for the treatment of various cancers, but a multitude of patients remain unresponsive for various reasons, including poor homing of T cells into tumors. In this study, we investigated the roles of the leukotriene B4 receptor, BLT1, and CXCR3, the receptor for CXCL9, CXCL10, and CXCL11, under endogenous as well as vaccine-induced antitumor immune response in a syngeneic murine model of B16 melanoma. Significant accelerations in tumor growth and reduced survival were observed in both BLT1(-/-) and CXCR3(-/-) mice as compared with wild-type (WT) mice. Analysis of tumor-infiltrating leukocytes revealed significant reduction of CD8(+) T cells in the tumors of BLT1(-/-) and CXCR3(-/-) mice as compared with WT tumors, despite their similar frequencies in the periphery. Adoptive transfer of WT but not BLT1(-/-) or CXCR3(-/-) CTLs significantly reduced tumor growth in Rag2(-/-) mice, a function attributed to reduced infiltration of knockout CTLs into tumors. Cotransfer experiments suggested that WT CTLs do not facilitate the infiltration of knockout CTLs to tumors. Anti-programmed cell death-1 (PD-1) treatment reduced the tumor growth rate in WT mice but not in BLT1(-/-), CXCR3(-/-), or BLT1(-/-)CXCR3(-/-) mice. The loss of efficacy correlated with failure of the knockout CTLs to infiltrate into tumors upon anti-PD-1 treatment, suggesting an obligate requirement for both BLT1 and CXCR3 in mediating anti-PD-1 based antitumor immune response. These results demonstrate a critical role for both BLT1 and CXCR3 in CTL migration to tumors and thus may be targeted to enhance efficacy of CTL-based immunotherapies.

Gut Microbiota Conversion of Dietary Ellagic Acid into Bioactive Phytoceutical Urolithin A Inhibits Heme Peroxidases.

Numerous studies signify that diets rich in phytochemicals offer many beneficial functions specifically during pathologic conditions, yet their effects are often not uniform due to inter-individual variation. The host indigenous gut microbiota and their modifications of dietary phytochemicals have emerged as factors that greatly influence the efficacy of phytoceutical-based intervention. Here, we investigated the biological activities of one such active microbial metabolite, Urolithin A (UA or 3,8-dihydroxybenzo[c]chromen-6-one), which is derived from the ellagic acid (EA). Our study demonstrates that UA potently inhibits heme peroxidases i.e. myeloperoxidase (MPO) and lactoperoxidase (LPO) when compared to the parent compound EA. In addition, chrome azurol S (CAS) assay suggests that EA, but not UA, is capable of binding to Fe3+, due to its catechol-like structure, although its modest heme peroxidase inhibitory activity is abrogated upon Fe3+-binding. Interestingly, UA-mediated MPO and LPO inhibition can be prevented by innate immune protein human NGAL or its murine ortholog lipocalin 2 (Lcn2), implying the complex nature of host innate immunity-microbiota interactions. Spectral analysis indicates that UA inhibits heme peroxidase-catalyzed reaction by reverting the peroxidase back to its inactive native state. In support of these in vitro results, UA significantly reduced phorbol myristate acetate (PMA)-induced superoxide generation in neutrophils, however, EA failed to block the superoxide generation. Treatment with UA significantly reduced PMA-induced mouse ear edema and MPO activity compared to EA treated mice. Collectively, our results demonstrate that microbiota-mediated conversion of EA to UA is advantageous to both host and microbiota i.e. UA-mediated inhibition of pro-oxidant enzymes reduce tissue inflammation, mitigate non-specific killing of gut bacteria, and abrogate iron-binding property of EA, thus providing a competitive edge to the microbiota in acquiring limiting nutrient iron and thrive in the gut.

Impact of Bi-Axial Shear on Atherogenic Gene Expression by Endothelial Cells.

This study demonstrated the effects of the directionality of oscillatory wall shear stress (WSS) on proliferation and proatherogenic gene expression (I-CAM, E-Selectin, and IL-6) in the presence of inflammatory mediators leukotriene B4 (LTB4) and bacterial lipopolysaccharide (LPS) from endothelial cells grown in an orbiting culture dish. Computational fluid dynamics (CFD) was applied to quantify the flow in the dish, while an analytical solution representing an extension of Stokes second problem was used for validation. Results indicated that WSS magnitude was relatively constant near the center of the dish and oscillated significantly (0-0.9 Pa) near the side walls. Experiments showed that LTB4 dominated the shear effects on cell proliferation and area. Addition of LPS didn't change proliferation, but significantly affected cell area. The expression of I-CAM1, E-Selectin and IL-6 were altered by directional oscillatory shear index (DOSI, a measure of the biaxiality of oscillatory shear), but not shear magnitude. The significance of DOSI was further reinforced by the strength of its interactions with other atherogenic factors. Hence, directionality of shear appears to be an important factor in regulating gene expression and provides a potential explanation of the propensity for increased vascular lesions in regions in the arteries with oscillating biaxial flow.

A spontaneous metastasis model reveals the significance of claudin-9 overexpression in lung cancer metastasis.

Metastasis causes most cancer related mortality but the mechanisms governing metastatic dissemination are poorly defined. Metastasis involves egression of cancer cells from the primary tumors, their survival in circulation and colonization at the secondary sites. Cancer cell egression from the primary tumor is the least defined process of metastasis as experimental metastasis models directly seed cancer cells in circulation, thus bypassing this crucial step. Here, we developed a spontaneous metastasis model that retains the egression step of metastasis. By repeated in vivo passaging of the poorly metastatic Lewis lung carcinoma (3LL) cells, we generated a cell line (p-3LL) that readily metastasizes to lungs and liver from subcutaneous (s.c.) tumors. Interestingly, when injected intravenously, 3LL and p-3LL cells showed a similar frequency of metastasis. This suggests enhanced egression of p-3LL cells may underlie the enhanced metastatic spread from primary tumors. Microarray analysis of 3LL and p-3LL cells as well as the primary tumors derived from these cells revealed altered expression of several genes including significant upregulation of a tight junction protein, claudin-9. Increased expression of claudin-9 was confirmed in both p-3LL cells and tumors derived from these cells. Knockdown of claudin-9 expression in p-3LL cells by si-RNA significantly reduced their motility, invasiveness in vitro and metastasis in vivo. Conversely, transient overexpression of claudin-9 in 3LL cells enhanced their motility. These results suggest an essential role for claudin-9 in promoting lung cancer metastasis.

Enterobacteria-secreted particles induce production of exosome-like S1P-containing particles by intestinal epithelium to drive Th17-mediated tumorigenesis.

Gut-associated inflammation plays a crucial role in the progression of colon cancer. Here, we identify a novel pathogen-host interaction that promotes gut inflammation and the development of colon cancer. We find that enteropathogenic bacteria-secreted particles (ET-BSPs) stimulate intestinal epithelium to produce IDENs (intestinal mucosa-derived exosome-like nanoparticles) containing elevated levels of sphingosine-1-phosphate, CCL20 and prostaglandin E2 (PGE2). CCL20 and PGE2 are required for the recruitment and proliferation, respectively, of Th17 cells, and these processes also involve the MyD88-mediated pathway. By influencing the recruitment and proliferation of Th17 cells in the intestine, IDENs promote colon cancer. We demonstrate the biological effect of sphingosine-1-phosphate contained in IDENs on tumour growth in spontaneous and transplanted colon cancer mouse models. These findings provide deeper insights into how host-microbe relationships are mediated by particles secreted from both bacterial and host cells.

Crystalline silica-induced leukotriene B4-dependent inflammation promotes lung tumour growth.

Chronic exposure to crystalline silica (CS) causes silicosis, an irreversible lung inflammatory disease that may eventually lead to lung cancer. In this study, we demonstrate that in K-ras(LA1) mice, CS exposure markedly enhances the lung tumour burden and genetic deletion of leukotriene B4 receptor-1 (BLT1(-/-)) attenuates this increase. Pulmonary neutrophilic inflammation induced by CS is significantly reduced in BLT1(-/-)K-ras(LA1) mice. CS exposure induces LTB4 production by mast cells and macrophages independent of inflammasome activation. In an air-pouch model, CS-induced neutrophil recruitment is dependent on LTB4 production by mast cells and BLT1 expression on neutrophils. In an implantable lung tumour model, CS exposure results in rapid tumour growth and decreased survival that is attenuated in the absence of BLT1. These results suggest that the LTB4/BLT1 axis sets the pace of CS-induced sterile inflammation that promotes lung cancer progression. This knowledge may facilitate development of immunotherapeutic strategies to fight silicosis and lung cancer.