Elizabeth A. Rondini

Vanadium pentoxide induces pulmonary inflammation and tumor promotion in a strain-dependent manner

BackgroundElevated levels of air pollution are associated with increased risk of lung cancer. Particulate matter (PM) contains transition metals that may potentiate neoplastic development through the induction of oxidative stress and inflammation, a lung cancer risk factor. Vanadium pentoxide (V2O5) is a component of PM derived from fuel combustion as well as a source of occupational exposure in humans. In the current investigation we examined the influence of genetic background on susceptibility to V2O5-induced inflammation and evaluated whether V2O5 functions as a tumor promoter using a 2-stage (initiation-promotion) model of pulmonary neoplasia in mice.ResultsA/J, BALB/cJ (BALB), and C57BL/6J (B6) mice were treated either with the initiator 3-methylcholanthrene (MCA; 10 μg/g; i.p.) or corn oil followed by 5 weekly aspirations of V2O5 or PBS and pulmonary tumors were enumerated 20 weeks following MCA treatment. Susceptibility to V2O5-induced pulmonary inflammation was assessed in bronchoalveolar lavage fluid (BALF), and chemokines, transcription factor activity, and MAPK signaling were quantified in lung homogenates. We found that treatment of animals with MCA followed by V2O5 promoted lung tumors in both A/J (10.3 ± 0.9 tumors/mouse) and BALB (2.2 ± 0.36) mice significantly above that observed with MCA/PBS or V2O5 alone (P < 0.05). No tumors were observed in the B6 mice in any of the experimental groups. Mice sensitive to tumor promotion by V2O5 were also found to be more susceptible to V2O5-induced pulmonary inflammation and hyperpermeability (A/J>BALB>B6). Differential strain responses in inflammation were positively associated with elevated levels of the chemokines KC and MCP-1, higher NFκB and c-Fos binding activity, as well as sustained ERK1/2 activation in lung tissue.ConclusionsIn this study we demonstrate that V2O5, an occupational and environmentally relevant metal oxide, functions as an in vivo lung tumor promoter among different inbred strains of mice. Further, we identified a positive relationship between tumor promotion and susceptibility to V2O5-induced pulmonary inflammation. These findings suggest that repeated exposures to V2O5 containing particles may augment lung carcinogenesis in susceptible individuals through oxidative stress mediated pathways.

Energy Balance Modulates Colon Tumor Growth: Interactive Roles of Insulin and Estrogen

Obesity increases colorectal cancer (CRC) risk and progression. However, the impact of obesity on CRC in women is dependent on ovarian hormone status. The purpose of this study was to determine the interactive roles of obesity and ovarian hormones on serum markers of inflammation, cell signaling, and transplanted colon tumor growth. Female C57BL/6 mice (6 wk) were either ovariectomized (OVX) or ovaries left intact (nonovariectomized, NOVX) and randomized to receive a (1) control, (2) 30% calorie‐restricted (CR), or (3) diet‐induced obese (DIO) diet regimen for 20 wk to induce differing levels of adiposity. Serum was collected and inflammatory and metabolic markers were measured using an antibody array (62 proteins) and ELISAs. Mice were subcutaneously injected with syngeneic MC38 colon cancer cells after 20 wk and sacrificed 4 wk later. CR mice had the smallest tumors irrespective of hormone status, whereas the largest tumors were observed in DIO‐OVX mice. Glucose tolerance was impaired in OVX mice, being most severe in the DIO‐OVX group. Cytokine arrays suggested that in CR animals, inhibition of tumor growth paralleled insulin sensitivity and associated changes in leptin, adiponectin, and IGF‐BPs. Conversely, in DIO‐OVX animals, tumor growth was associated with insulin and leptin resistance as well as higher levels of pro‐inflammatory proteins. In vitro, leptin and adiponectin had no effect, whereas insulin induced MC38 cell proliferation and MAPK activation. Co‐treatment with estrogen blocked the stimulatory effects of insulin. Thus, our in vitro and in vivo data indicate female reproductive hormones have a modulating effect on obesity‐induced insulin resistance and inflammation, which may directly or indirectly influence CRC progression. ©2010 Wiley‐Liss, Inc.

Supplementation with Galacto-Oligosaccharides Increases the Percentage of NK Cells and Reduces Colitis Severity in Smad3-Deficient Mice

The gut microbiota plays an essential role in intestinal immunity. Prebiotics, including galacto-oligosaccharides (GOS), are fermentable fibers that beneficially affect the host by stimulating the growth of specific microbial populations. We investigated the effect of GOS on colitis development and on immune variables in Smad3-deficient mice treated with the pathogen Helicobacter hepaticus. Mice were supplemented daily with 5000 mg GOS/kg body weight 2 wk prior to infection and 4 wk postinfection, a time period during which colitis severity peaks in this model. Mice (n = 4-8/treatment at each time) were killed preinfection (0 d) and at 3, 7, and 28 d postinfection to evaluate immune variables in the spleen and in mesenteric lymph nodes (MsLN) by flow cytometry. Colon and cecum samples were collected for histopathologic analysis. Fecal pellets (n = 8-9/treatment) were collected prior to infection to measure relative changes in Bifidobacterium ssp. and Lactobacillum ssp. by real-time PCR. GOS significantly reduced colitis severity in response to H. hepaticus (P < 0.0001). This was associated with a significant increase in the percentage of NK cells in the spleen (P < 0.001) and in MsLN (P < 0.001) at 3 d postinfection and a 1.5-fold increase in fecal Bifidobacterium ssp. (P = 0.003). GOS stimulated NK expression of CCR9, a chemokine receptor involved in lymphocyte trafficking to the gut preinfection (0 d) in the blood (P = 0.02), spleen (P = 0.033), and MsLN (P = 0.017). In addition, GOS stimulated colonic IL-15 production 3 d postinfection (P < 0.001). These data suggest that GOS reduces colitis by modulating the function and trafficking of NK cells and may provide a novel therapeutic strategy for individuals with inflammatory bowel disease.

Identification of candidate genes downstream of TLR4 signaling after ozone exposure in mice: A role for heat-shock protein 70

Background: Toll-like receptor 4 (TLR4) is involved in ozone (O3)-induced pulmonary hyperpermeability and inflammation, although the downstream signaling events are unknown. Objectives: The aims of our study were to determine the mechanism through which TLR4 modulates O3-induced pulmonary responses and to use transcriptomics to determine potential TLR4 effector molecules. Methods: C3H/HeJ (HeJ; Tlr4 mutant) and C3H/HeOuJ (OuJ; Tlr4 normal) mice were exposed continuously to 0.3 ppm O3 or filtered air for 6, 24, 48, or 72 hr. We assessed inflammation using bronchoalveolar lavage and molecular analysis by mRNA microarray, quantitative RT-PCR (real-time polymerase chain reaction), immunoblots, immunostaining, and ELISAs (enzyme-linked immunosorbent assays). B6-Hspa1a/Hspa1btm1Dix/NIEHS (Hsp70–/–) and C57BL/6 (B6; Hsp70+/+ wild-type control) mice were used for candidate gene validation studies. Results: O3-induced TLR4 signaling occurred through myeloid differentiation protein 88 (MyD88)-dependent and -independent pathways in OuJ mice and involved multiple downstream pathways. Genomewide transcript analyses of lungs from air- and O3-exposed HeJ and OuJ mice identified a cluster of genes that were significantly up-regulated in O3-exposed OuJ mice compared with O3-exposed HeJ mice or air-exposed controls of both strains; this cluster included genes for heat-shock proteins (e.g., Hspa1b, Hsp70). Moreover, O3-induced inflammation, MyD88 
up-regulation, extracellular-signal–related kinase-1/2 (ERK1/2) and activator protein-1 (AP-1) activation, and kerotinocyte-derived chemokine (KC) protein content were significantly reduced in Hspa1a/Hspa1btm1Dix (Hsp70–/–) compared with Hsp70+/+ mice (p < 0.05). Conclusions: These studies suggest that HSP70 is an effector molecule downstream of TLR4 and is involved in the regulation of O3-induced lung inflammation by triggering similar pathways to TLR4. These novel findings may have therapeutic and preventive implications for inflammatory diseases resulting from environmental exposures.

Identification of novel susceptibility genes in ozone-induced inflammation in mice

Ozone (O3) remains a prevalent air pollutant and public health concern. Inf2 is a significant quantitative trait locus on murine chromosome 17 that contributes to susceptibility to O3-induced infiltration of polymorphonuclear leukocytes (PMNs) into the lung, but the mechanisms of susceptibility remain unclear. The study objectives were to confirm and restrict Inf2, and to identify and test novel candidate susceptibility gene(s). Congenic strains of mice that contained overlapping regions of Inf2 and their controls, and mice deficient in either major histocompatibility complex (MHC) class II genes or the Tnf cluster, were exposed to air or O3. Lung inflammation and gene expression were assessed. Inf2 was restricted from 16.42 Mbp to 0.96 Mbp, and bioinformatic analysis identified MHC class II, the Tnf cluster and other genes in this region that contain potentially informative single nucleotide polymorphisms between the susceptible and resistant mice. Furthermore, O3-induced inflammation was significantly reduced in mice deficient in MHC class II genes or the Tnf cluster genes, compared with wild-type controls. Gene expression differences were also observed in MHC class II and Tnf cluster genes. This integrative genetic analysis of Inf2 led to identification of novel O3 susceptibility genes that may provide important, new therapeutic targets in susceptible individuals.

Differential effects of energy balance on experimentally-induced colitis

AIM To characterize the influence of diet-induced changes in body fat on colitis severity in SMAD3-/- mice. METHODS SMAD3-/- mice (6-8 wk of age) were randomly assigned to receive a calorie restricted (30% of control; CR), control (CON), or high fat (HF) diet for 20 wk and were gavaged with sterile broth or with Helicobacter hepaticus (H. hepaticus) to induce colitis. Four weeks after infection, mice were sacrificed and the cecum and colons were processed for histological evaluation. RESULTS Dietary treatment significantly influenced body composition prior to infection (P < 0.05), with CR mice having less (14% ± 2%) and HF-fed mice more body fat (32% ± 7%) compared to controls (22% ± 4%). Differences in body composition were associated with alterations in plasma levels of leptin (HF > CON > CR) and adiponectin (CON > HF ≥ CR) (P < 0.05). There were no significant differences in colitis scores between CON and HF-fed mice 4 wk post-infection. Consistent with this, differences in proliferation and inflammation markers (COX-2, iNOS), and infiltrating cell types (CD3+ T lymphocytes, macrophages) were not observed. Unexpectedly, only 40% of CR mice survived infection with H. hepaticus, with mortality observed as early as 1 wk following induction of colitis. CONCLUSION Increased adiposity does not influence colitis severity in SMAD3-/- mice. Importantly, caloric restriction negatively impacts survival following pathogen challenge, potentially due to an impaired immune response.

Microarray Analyses of Genes Differentially Expressed by Diet (Black Beans and Soy Flour) during Azoxymethane-Induced Colon Carcinogenesis in Rats

We previously demonstrated that black bean (BB) and soy flour (SF)-based diets inhibit azoxymethane (AOM)-induced colon cancer. The objective of this study was to identify genes altered by carcinogen treatment in normal-appearing colonic mucosa and those attenuated by bean feeding. Ninety-five male F344 rats were fed control (AIN) diets upon arrival. At 4 and 5 weeks, rats were injected with AOM (15 mg/kg) or saline and one week later administered an AIN, BB-, or SF-based diet. Rats were sacrificed after 31 weeks, and microarrays were conducted on RNA isolated from the distal colonic mucosa. AOM treatment induced a number of genes involved in immunity, including several MHC II-associated antigens and innate defense genes (RatNP-3, Lyz2, Pla2g2a). BB- and SF-fed rats exhibited a higher expression of genes involved in energy metabolism and water and sodium absorption and lower expression of innate (RatNP-3, Pla2g2a, Tlr4, Dmbt1) and cell cycle-associated (Cdc2, Ccnb1, Top2a) genes. Genes involved in the extracellular matrix (Col1a1, Fn1) and innate immunity (RatNP-3, Pla2g2a) were induced by AOM in all diets, but to a lower extent in bean-fed animals. This profile suggests beans inhibit colon carcinogenesis by modulating cellular kinetics and reducing inflammation, potentially by preserving mucosal barrier function.

Increased presence of effector lymphocytes during Helicobacter hepaticus -induced colitis

AIM To identify and characterize drosophila mothers against decapentaplegic (SMAD)3-dependent changes in immune cell populations following infection with Helicobacter hepaticus (H. hepaticus). METHODS SMAD3(-/-) (n = 19) and colitis-resistant SMAD3(+/-) (n = 24) mice (8-10 wk of age) were infected with H. hepaticus and changes in immune cell populations [T lymphocytes, natural killer (NK) cells, T regulatory cells] were measured in the spleen and mesenteric lymph nodes (MsLNs) at 0 d, 3 d, 7 d and 28 d post-infection using flow cytometry. Genotype-dependent changes in T lymphocytes and granzyme B(+) cells were also assessed after 28 d in proximal colon tissue using immunohistochemistry. RESULTS As previously observed, SMAD3(-/-), but not SMAD3(+/-) mice, developed colitis, peaking at 4 wk post-infection. No significant changes in T cell subsets were observed in the spleen or in the MsLNs between genotypes at any time point. However, CD4(+) and CD8(+)/CD62L(lo) cells, an effector T lymphocyte population, as well as NK cells (NKp46/DX5(+)) were significantly higher in the MsLNs of SMAD3(-/-) mice at 7 d and 28 d post-infection. In the colon, a higher number of CD3(+) cells were present in SMAD3(-/-) compared to SMAD3(+/-) mice at baseline, which did not significantly change during infection. However, the number of granzyme B(+) cells, a marker of cytolytic lymphocytes, significantly increased in SMAD3(-/-) mice 28 d post-infection compared to both SMAD3(+/-) mice and to baseline values. This was consistent with more severe colitis development in these animals. CONCLUSION Data suggest that defects in SMAD3 signaling increase susceptibility to H. hepaticus-induced colitis through aberrant activation and/or dysregulation of effector lymphocytes.

Transforming growth factor β2 is negatively regulated by endogenous retinoic acid during early heart morphogenesis

Vitamin A‐deficient (VAD) quail embryos lack the vitamin A‐active form, retinoic acid (RA) and are characterized by a phenotype that includes a grossly abnormal cardiovascular system that can be rescued by RA. Here we report that the transforming growth factor, TGFβ2 is involved in RA‐regulated cardiovascular development. In VAD embryos TGFβ2 mRNA and protein expression are greatly elevated. The expression of TGFβ receptor II is also elevated in VAD embryos but is normalized by treatment with TGFβ2‐specific antisense oligonucleotides (AS). Administration of this AS or an antibody specific for TGFβ2 to VAD embryos normalizes posterior heart development and vascularization, while the administration of exogenous active TGFβ2 protein to normal quail embryos mimics the excessive TGFβ2 status of VAD embryos and induces VAD cardiovascular phenotype. In VAD embryos pSmad2/3 and pErk1 are not activated, while pErk2 and pcRaf are elevated and pSmad1/5/8 is diminished. We conclude that in the early avian embryo TGFβ2 has a major role in the retinoic acid‐regulated posterior heart morphogenesis for which it does not use Smad2/3 pathways, but may use other signaling pathways. Importantly, we conclude that retinoic acid is a critical negative physiological regulator of the magnitude of TGFβ2 signals during vertebrate heart formation.

Toll-like receptor expression in human non-small cell lung carcinoma: potential prognostic indicators of disease

Introduction Lung cancer remains the highest cause of cancer mortality worldwide. Toll-like receptors (TLR) are innate immune receptors that have both pro- and anti-tumorigenic properties. Based on findings from epidemiological studies and in rodents, we hypothesized that elevated TLR expression would be a positive prognostic indicator of disease in non-small cell lung carcinoma patients. Results Higher mRNA expression of TLR1-3 and 5-8 were significantly associated with increased overall survival (OS) when analyzed individually or as a group in both non-small cell lung carcinoma (NSCLC) patients and in the adenocarcinoma (ADC) subtype. Significant co-expression of many TLR combinations in ADC patients were also observed via RNA sequencing. Immunostaining demonstrated TLR4 and 8 significantly correlated in tumor tissue, similar to RNA. Methods We used kmplot.com to perform a meta-analysis on mRNA expression of TLR1-10 to determine any significant associations with OS in NSCLC and the ADC subtype. cBioportal was also used simultaneously to assess co-expression in TLR1-10 in ADC patients via RNA sequencing and to identify any molecular alterations. Lastly, immunostaining for a subset of TLRs was conducted on ADC patients. Conclusions Expression of innate immune receptors TLR1-10 is associated with improved survival outcomes in NSCLC. Thus, further evaluation of their predictive capacity and therapeutic utility is warranted.INTRODUCTION Lung cancer remains the highest cause of cancer mortality worldwide. Toll-like receptors (TLR) are innate immune receptors that have both pro- and anti-tumorigenic properties. Based on findings from epidemiological studies and in rodents, we hypothesized that elevated TLR expression would be a positive prognostic indicator of disease in non-small cell lung carcinoma patients. RESULTS Higher mRNA expression of TLR1-3 and 5-8 were significantly associated with increased overall survival (OS) when analyzed individually or as a group in both non-small cell lung carcinoma (NSCLC) patients and in the adenocarcinoma (ADC) subtype. Significant co-expression of many TLR combinations in ADC patients were also observed via RNA sequencing. Immunostaining demonstrated TLR4 and 8 significantly correlated in tumor tissue, similar to RNA. METHODS We used kmplot.com to perform a meta-analysis on mRNA expression of TLR1-10 to determine any significant associations with OS in NSCLC and the ADC subtype. cBioportal was also used simultaneously to assess co-expression in TLR1-10 in ADC patients via RNA sequencing and to identify any molecular alterations. Lastly, immunostaining for a subset of TLRs was conducted on ADC patients. CONCLUSIONS Expression of innate immune receptors TLR1-10 is associated with improved survival outcomes in NSCLC. Thus, further evaluation of their predictive capacity and therapeutic utility is warranted.