AIM: To investigate differences in microbes and short chain fatty acid (SCFA) levels in stool samples from Hispanic and non-Hispanic African American, American Indian, and White participants.
METHODS: Stool samples from twenty participants were subjected to analysis for relative levels of viable bacteria and for SCFA levels. Additionally, the samples were subjected to 16S rRNA gene pyrosequencing for identification of bacteria present in the stool. We used a metagenome functional prediction technique to analyze genome copy numbers and estimate the abundance of butyrate kinase in all samples.
RESULTS: We found that African Americans had significantly lower levels of acetate, butyrate, and total SCFAs than all other racial/ethnic groups. We also found that participant microbial profiles differed by racial/ethnic group. African Americans had significantly more Firmicutes than Whites, with enriched Ruminococcaceae. The Firmicutes/Bacteroidetes ratio was also significantly higher for African Americans than for Whites (P = 0.049). We found Clostridium levels to be significantly and inversely related to total SCFA levels (P = 0.019) and we found Bacteroides to be positively associated (P = 0.027) and Clostridium to be negatively associated (P = 0.012) with levels of butyrate. We also identified a correlation between copy number for a butyrate kinase predicted from 16S rRNA gene abundance and levels of butyrate in stool.
CONCLUSION: The identified differences in gut flora and SCFA levels may relate to colorectal cancer mortality differentials and may be useful as targets for future clinical and behavioral interventions.
Colorectal cancer; Short chain fatty acids; Racial/ethnic disparities; Butyrate; Microbiota
Leukotriene B4 (LTB4) receptor, BLT1 is expressed on variety of immune cells and has been implicated as a mediator of diverse inflammatory diseases. However, whether biological responses initiated via this receptor generate tumor promoting inflammation or anti-tumor immunity remains unexplored. In this study, we investigated the role of BLT1 in antitumor immunity using syngeneic TC-1 cervical cancer model and observed accelerated tumor growth and reduced survival in BLT1−/− mice compared to BLT1+/+ mice. Analysis of the tumor infiltrates by flow cytometry and confocal microscopy revealed a significant decrease in effector immune cells, most notably CD8+-T cells and NK cells in the tumors of the BLT1−/− mice. Gene expression profiling confirmed the dramatic decrease of IFN-γ, granzyme-B and IL-2 in tumors growing in BLT1−/− mice. Furthermore, depletion of CD8+ T cells enhanced the tumor growth in BLT1+/+ but not in BLT1−/− mice. However, similar levels of antigen dependent CD8+ T cell mediated killing activity were observed in spleens of BLT1+/+ and BLT1−/− mice. Adoptive transfer of CD8+ T cells from tumor bearing BLT1+/+ but not BLT1−/− mice significantly reduced tumor growth and increased the survival of Rag2−/− mice. While the homeostatic proliferation and expression profiles of other chemokine receptors of adoptively transferred BLT1+/+ and BLT1−/− CD8+ T cells appears to be similar, BLT1+/+ T-lymphocytes entered the tumors in greater numbers. These results suggest that BLT1 expression on CD8+ T cells plays an important role in their trafficking to tumors.
Leukotriene B4; BLT1; cancer; anti-tumor immunity; leukocyte migration
Cryptosporidium parasites are a major cause of diarrhea and malnutrition in the developing world, a frequent cause of waterborne disease in the developed world, and a potential bioterrorism agent. Currently, available treatment is limited, and Cryptosporidium drug discovery remains largely unsuccessful. As a result, the pharmacokinetic properties required for in vivo efficacy have not been established. We have been engaged in a Cryptosporidium drug discovery program targeting IMP dehydrogenase (CpIMPDH). Here, we report the activity of eight potent and selective inhibitors of CpIMPDH in the interleukin-12 (IL-12) knockout mouse model, which mimics acute human cryptosporidiosis. Two compounds displayed significant antiparasitic activity, validating CpIMPDH as a drug target. The best compound, P131 (250 mg/kg of body weight/day), performed equivalently to paromomycin (2,000 mg/kg/day) when administered in a single dose and better than paromomycin when administered in three daily doses. One compound, A110, appeared to promote Cryptosporidium infection. The pharmacokinetic, uptake, and permeability properties of the eight compounds were measured. P131 had the lowest systemic distribution but accumulated to high concentrations within intestinal cells. A110 had the highest systemic distribution. These observations suggest that systemic distribution is not required, and may be a liability, for in vivo antiparasitic activity. Intriguingly, A110 caused specific alterations in fecal microbiota that were not observed with P131 or vehicle alone. Such changes may explain how A110 promotes parasitemia. Collectively, these observations suggest a blueprint for the development of anticryptosporidial therapy.
G-protein-coupled estrogen receptor (GPER/GPR30) was reported to bind 17β-estradiol (E2), tamoxifen, and ICI 182,780 (fulvestrant) and promotes activation of epidermal growth factor receptor (EGFR)-mediated signaling in breast, endometrial and thyroid cancer cells. Although lung adenocarcinomas express estrogen receptors α and β (ERα and ERβ), the expression of GPER in lung cancer has not been investigated. The purpose of this study was to examine the expression of GPER in lung cancer.
The expression patterns of GPER in various lung cancer lines and lung tumors were investigated using standard quantitative real time PCR (at mRNA levels), Western blot and immunohistochemistry (IHC) methods (at protein levels). The expression of GPER was scored and the pairwise comparisons (cancer vs adjacent tissues as well as cancer vs normal lung tissues) were performed.
Analysis by real-time PCR and Western blotting revealed a significantly higher expression of GPER at both mRNA and protein levels in human non small cell lung cancer cell (NSCLC) lines relative to immortalized normal lung bronchial epithelial cells (HBECs). The virally immortalized human small airway epithelial cell line HPL1D showed higher expression than HBECs and similar expression to NSCLC cells. Immunohistochemical analysis of tissue sections of murine lung adenomas as well as human lung adenocarcinomas, squamous cell carcinomas and non-small cell lung carcinomas showed consistently higher expression of GPER in the tumor relative to the surrounding non-tumor tissue.
The results from this study demonstrate increased GPER expression in lung cancer cells and tumors compared to normal lung. Further evaluation of the function and regulation of GPER will be necessary to determine if GPER is a marker of lung cancer progression.
GPER; GPR30; Estrogen; Estrogen receptor; Lung cancer; Protein expression; Immunohistochemistry; Tissue microarray
Chronic inflammation is an underlying factor linking obesity with insulin resistance. Diet-induced obesity promotes an increase in circulating levels of inflammatory monocytes and their infiltration into expanding adipose tissue. Nevertheless, the endogenous pathways that trigger and sustain chronic low-grade inflammation in obesity are incompletely understood. Here, we report that a high-fat diet selectively increases the circulating levels of CD11b+ monocytes in wild-type mice that express leukotriene B4 receptor, BLT-1, and that this increase is abolished in BLT-1-null mice. The accumulation of classically activated (M1) adipose tissue macrophages (ATMs) and the expression of pro-inflammatory cytokines and chemokines (i.e. IL-6 and Ccl2) was largely blunted in adipose tissue of obese BLT-1-/- mice, while the ratio of alternatively activated (M2) ATMs to M1 ATMs was increased. Obese BLT-1-/- mice were protected from systemic glucose and insulin intolerance and this was associated with a decrease in inflammation in adipose tissue and liver and a decrease in hepatic triglyceride accumulation. Deletion of BLT-1 prevented high-fat induced loss of insulin signaling in liver and skeletal muscle. These observations elucidate a novel role of chemoattractant receptor, BLT-1, in promoting monocyte trafficking to adipose tissue and promoting chronic inflammation in obesity and could lead to the identification of new therapeutic targets for treating insulin resistance in obesity.
Rationale: Obstructive sleep apnea, which is characterized by intermittent hypoxia (IH) during sleep, has emerged as an independent risk factor for cardiovascular disease, including atherosclerosis. Leukotriene B4 (LTB4) production is increased in patients with obstructive sleep apnea and negatively correlates to hypoxic levels during sleep, with continuous positive airway pressure therapy decreasing LTB4 production.
Objectives: Determine the potential role of LTB4 in IH-induced atherosclerosis in a monocyte cellular model and a murine model.
Methods: THP-1 cells were exposed to IH for 3, 6, 24, and 48 hours. Macrophage transformation and foam cell formation were assessed after IH exposures. Apolipopotein E (ApoE)−/− or BLT1−/−/ApoE−/− mice were fed an atherogenic diet and exposed to IH (alternating 21% and 5.7% O2 from 7 am to 7 PM each day) for 10 weeks. Atherosclerotic lesion formation in en face aorta was examined by oil red O staining.
Measurements and Main Results: IH increased production of LTB4 and the expression of 5-lipoxygenase and leukotriene A4 hydrolase, the key enzymes for producing LTB4. IH was associated with transformation of monocytes to activated macrophages, as evidenced by increased expression of CD14 and CD68. In addition, IH exposures promoted increased cellular cholesterol accumulation and foam cell formation. The LTB4 receptor 1 (BLT1) antagonist U-75302 markedly attenuated IH-induced changes. Furthermore, IH promoted atherosclerotic lesion formation in ApoE−/− mice. IH-induced lesion formation was markedly attenuated in BLT1−/−/ApoE−/− mice.
Conclusions: BLT1-dependent pathways underlie IH-induced atherogenesis, and may become a potential novel therapeutic target for obstructive sleep apnea–associated cardiovascular disease.
obstructive sleep apnea; inflammation; monocyte; atherosclerosis
A large and diverse array of chemoattractants control leukocyte trafficking, but how these apparently redundant signals collaborate in vivo is still largely unknown. We previously demonstrated an absolute requirement for the lipid chemoattractant leukotriene B4 (LTB4) and its receptor BLT1 for neutrophil recruitment into the joint in autoantibody-induced arthritis. We now demonstrate that BLT1 is required for neutrophils to deliver IL-1 into the joint to initiate arthritis. IL-1-expressing neutrophils amplify arthritis through the production of neutrophil-active chemokines from synovial tissue cells. CCR1 and CXCR2, two neutrophil chemokine receptors, operate non-redundantly to sequentially control the later phase of neutrophil recruitment into the joint and mediate all neutrophil chemokine activity in the model. Thus, we have uncovered a complex sequential relationship involving unique contributions from the lipid mediator LTB4, the cytokine IL-1, and CCR1 and CXCR2 chemokine ligands that are all absolutely required for effective neutrophil recruitment into the joint.
Sphingosine-1-phosphate (S1P) receptor subtype 1 (S1P1), a G-protein coupled receptor (GPCR), regulates many biological activities of endothelial cells (ECs). In this report, we show that S1P1 receptors are present in the nuclei of ECs by using various biochemical and microscopic techniques such as cellular fractionation, immunogold labeling, and confocal microscopic analysis. Live cell imaging showed that plasma membrane S1P1 receptors are rapidly internalized and subsequently translocated to nuclear compartment upon S1P stimulation. Utilizing membrane biotinylation technique further supports the notion that nuclear S1P1 receptors were internalized from plasma membrane S1P1 after ligand treatment. Moreover, nuclear S1P1 is able to regulate the transcription of Cyr61 and CTGF, two growth factors functionally important in the regulation of vasculature. Collectively, these data suggest a novel S1P–S1P1 signaling axis present in the nuclear compartment of endothelial cells, which may regulate biological responses of endothelium.
S1P; S1P1; Endothelial cells
The somatic mutation hypothesis of cancer predicts that reducing the frequency of mutations induced by carcinogens will reduce the incidence of cancer. To examine this, we developed an antimutator strategy based on the manipulation of the level of a protein required for mutagenic bypass of DNA damage induced by the ubiquitous carcinogen benzo[a]pyrene. The expression of this protein, REV1, was reduced in mouse cells using a vector encoding a gene-specific targeting ribozyme. In the latter cells, mutagenesis induced by the activated form of benzo[a]pyrene was reduced over 90%. To examine if REV1 transcripts could be lowered in vivo, the plasmid was complexed with polyethyleneimine, a non-viral cationic polymer, and delivered to the lung via aerosol. The endogenous REV1 transcript in the bronchial epithelium as determined by quantitative real-time PCR in laser capture microdissected cells was reduced by 60%. There was a significant decrease in the multiplicity of carcinogen induced lung tumors from 6.4 tumors/ mouse to 3.7 tumors/ mouse. Additionally, REV1 inhibition completely abolished tumor formation in 27% of the carcinogen-exposed mice. These data support the central role of the translesion synthesis pathway in the development of lung cancer. Further, the selective modulation of members of this pathway presents novel potential targets for cancer prevention.
REV1; benzo[a]pyrene; mutagenesis; lung cancer; translesion synthesis
The purpose of this review is to summarize the role that murine models of arthritis are playing in the understanding of human rheumatoid arthritis and how leukotriene B4 (LTB4) is emerging as an important target in this field. Both the collagen-induced arthritis (CIA) model and the K/BxN serum transfer arthritis model have contributed to outline the potential mechanisms involved in inflammatory arthritis. Indeed, the CIA model has contributed to the development of effective anti-TNF and anti-IL-1β based treatments for RA that are currently in the clinic. Many recent studies in mouse models have suggested a critical role for LTB4 and its receptors in the development of inflammatory arthritis. Inhibitors of LTB4 biosynthesis as well as LTB4 receptors are protective in mouse models of RA and mice deficient in the LTB4 biosynthetic enzymes or LTB4 receptors are resistant to disease development suggesting several promising targets for RA in this pathway.
Rheumatoid Arthritis; Leukotriene B4 receptors; arthritis mouse models; FLAP
Rationale: Leukotriene B4 (LTB4) is a rapidly synthesized, early leukocyte chemoattractant that signals via its cell surface receptor, leukotriene B4 receptor 1 (BLT1), to attract and activate leukocytes during inflammation. A role for the LTB4–BLT1 pathway in allergen-induced airway hyperresponsiveness and inflammation is not well defined. Objectives: To define the role of the LTB4 receptor (BLT1) in the development of airway inflammation and altered airway function. Methods: BLT1-deficient (BLT1−/−) mice and wild-type mice were sensitized to ovalbumin by intraperitoneal injection and then challenged with ovalbumin via the airways. Airway responsiveness to inhaled methacholine, bronchoalveolar lavage fluid cell composition and cytokine levels, and lung inflammation and goblet cell hyperplasia were assessed. Results: Compared with wild-type mice, BLT1−/− mice developed significantly lower airway responsiveness to inhaled methacholine, lower goblet cell hyperplasia in the airways, and decreased interleukin (IL)-13 production both in vivo, in the bronchoalveolar lavage fluid, and in vitro, after antigen stimulation of lung cells in culture. Intracellular cytokine staining of lung cells revealed that bronchoalveolar lavage IL-13 levels and numbers of IL-13+/CD4+ and IL-13+/CD8+ T cells were also reduced in BLT1−/− mice. Reconstitution of sensitized and challenged BLT1−/− mice with allergen-sensitized BLT1+/+ T cells fully restored the development of airway hyperresponsiveness. In contrast, transfer of naive T cells failed to do so. Conclusion: These data suggest that BLT1 expression on primed T cells is required for the full development of airway hyperresponsiveness, which appears to be associated with IL-13 production in these cells.
airway responsiveness; cytokines; lipid mediators; lung inflammation; T cells
Leukotriene B4 (LTB4) is a rapidly synthesized, early neutrophil chemoattractant that signals via its cell surface receptor, BLT-1, to attract and activate neutrophils during peritonitis. BLT-1-deficient (BLT-1−/−) mice were used to determine the effects of LTB4 on neutrophil migration and activation, bacterial levels, and survival after cecal ligation and puncture (CLP). Male BLT-1−/− or wild-type (WT) BALB/c mice underwent CLP. Tissues were harvested for determination of levels of bacteria, myeloperoxidase (MPO), LTB4, macrophage inflammatory protein 2 (MIP-2), and neutrophil (polymorphonuclear leukocyte [PMN]) numbers at 4 and 18 h after CLP. PMN activation was determined by an assessment of phagocytosis ability and CD11b expression. Survival was also determined. BLT-1−/− mice had decreased numbers of PMNs in the peritoneum at both 4 and 18 h after CLP but increased numbers of PMNs in the blood at 18 h compared with WT mice. Liver and lung MPO levels were significantly higher in BLT-1−/− mice at both 4 and 18 h after CLP, with increased bacterial levels in the blood, the liver, and peritoneal fluid at 4 h. Bacterial levels remained higher in peritoneal fluid at 18 h, but blood and liver bacterial levels at 18 h were not different from levels at 4 h. PMN phagocytosis and CD11b levels were decreased in BLT-1−/− mice. LTB4 levels were similar between the groups before and after CLP, but MIP-2 levels were decreased both locally and systemically in BLT-1−/− mice. Survival was significantly improved in BLT-1−/− mice (71%) compared with WT mice (14%) at 48 h post-CLP. Thus, LTB4 modulates neutrophil migration into the mouse peritoneum, but not the lung or liver, after CLP. Despite higher bacterial and PMN levels at remote sites, there was increased survival in BLT-1−/− mice compared to WT mice. Decreased PMN activation may result in less remote organ dysfunction and improved survival.
Leukotrienes are derived from arachidonic acid and serve as mediators of inflammation and immediate hypersensitivity. Leukotriene B4 (LTB4) and leukotriene C4 (LTC4) act through G protein–coupled receptors LTB4 receptor (BLTR) and Cys-LTR, respectively. To investigate the physiological role of BLTR, we produced mice with a targeted disruption of the BLTR gene. Mice deficient for BLTR (BLTR−/−) developed normally and had no apparent hematopoietic abnormalities. Peritoneal neutrophils from BLTR−/− mice displayed normal responses to the inflammatory mediators C5a and platelet-activating factor (PAF) but did not respond to LTB4 for calcium mobilization or chemotaxis. Additionally, LTB4 elicited peritoneal neutrophil influx in control but not in BLTR−/− mice. Thus, BLTR is the sole receptor for LTB4-induced inflammation in mice. Neutrophil influx in a peritonitis model and acute ear inflammation in response to arachidonic acid was significantly reduced in BLTR−/− mice. In mice, intravenous administration of PAF induces immediate lethal anaphylaxis. Surprisingly, female BLTR−/− mice displayed selective survival (6 of 9; P = 0.002) relative to male (1 of 11) mice of PAF-induced anaphylaxis. These results demonstrate the role of BLTR in leukotriene-mediated acute inflammation and an unexpected sex-related involvement in PAF-induced anaphylaxis.
arachidonic acid; neutrophil influx; knock-out; sex-related; chemotaxis