Saracatinib, a Selective Src Kinase Inhibitor, Blocks Fibrotic Responses in Preclinical Models of Pulmonary Fibrosis.

Rationale: Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and often fatal disorder. Two U.S. Food and Drug Administration-approved antifibrotic drugs, nintedanib and pirfenidone, slow the rate of decline in lung function, but responses are variable and side effects are common. Objectives: Using an in silico data-driven approach, we identified a robust connection between the transcriptomic perturbations in IPF disease and those induced by saracatinib, a selective Src kinase inhibitor originally developed for oncological indications. Based on these observations, we hypothesized that saracatinib would be effective at attenuating pulmonary fibrosis. Methods: We investigated the antifibrotic efficacy of saracatinib relative to nintedanib and pirfenidone in three preclinical models: 1) in vitro in normal human lung fibroblasts; 2) in vivo in bleomycin and recombinant Ad-TGF-ß (adenovirus transforming growth factor-ß) murine models of pulmonary fibrosis; and 3) ex vivo in mice and human precision-cut lung slices from these two murine models as well as patients with IPF and healthy donors. Measurements and Main Results: In each model, the effectiveness of saracatinib in blocking fibrogenic responses was equal or superior to nintedanib and pirfenidone. Transcriptomic analyses of TGF-ß-stimulated normal human lung fibroblasts identified specific gene sets associated with fibrosis, including epithelial-mesenchymal transition, TGF-ß, and WNT signaling that was uniquely altered by saracatinib. Transcriptomic analysis of whole-lung extracts from the two animal models of pulmonary fibrosis revealed that saracatinib reverted many fibrogenic pathways, including epithelial-mesenchymal transition, immune responses, and extracellular matrix organization. Amelioration of fibrosis and inflammatory cascades in human precision-cut lung slices confirmed the potential therapeutic efficacy of saracatinib in human lung fibrosis. Conclusions: These studies identify novel Src-dependent fibrogenic pathways and support the study of the therapeutic effectiveness of saracatinib in IPF treatment.

Cutting Edge: IL-36 Receptor Promotes Resolution of Intestinal Damage.

IL-1 family members are central mediators of host defense. In this article, we show that the novel IL-1 family member IL-36γ was expressed during experimental colitis and human inflammatory bowel disease. Germ-free mice failed to induce IL-36γ in response to dextran sodium sulfate (DSS)-induced damage, suggesting that gut microbiota are involved in its induction. Surprisingly, IL-36R-deficient (Il1rl2(-/-)) mice exhibited defective recovery following DSS-induced damage and impaired closure of colonic mucosal biopsy wounds, which coincided with impaired neutrophil accumulation in the wound bed. Failure of Il1rl2(-/-) mice to recover from DSS-induced damage was associated with a profound reduction in IL-22 expression, particularly by colonic neutrophils. Defective recovery of Il1rl2(-/-) mice could be rescued by an aryl hydrocarbon receptor agonist, which was sufficient to restore IL-22 expression and promote full recovery from DSS-induced damage. These findings implicate the IL-36/IL-36R axis in the resolution of intestinal mucosal wounds.

A Wipe-Based Stool Collection and Preservation Kit for Microbiome Community Profiling.

While a range of methods for stool collection exist, many require complicated, self-directed protocols and stool transfer. In this study, we introduce and validate a novel, wipe-based approach to fecal sample collection and stabilization for metagenomics analysis. A total of 72 samples were collected across four different preservation types: freezing at -20°C, room temperature storage, a commercial DNA preservation kit, and a dissolvable wipe used with DESS (dimethyl sulfoxide, ethylenediaminetetraacetic acid, sodium chloride) solution. These samples were sequenced and analyzed for taxonomic abundance metrics, bacterial metabolic pathway classification, and diversity analysis. Overall, the DESS wipe results validated the use of a wipe-based capture method to collect stool samples for microbiome analysis, showing an R2 of 0.96 for species across all kingdoms, as well as exhibiting a maintenance of Shannon diversity (3.1-3.3) and species richness (151-159) compared to frozen samples. Moreover, DESS showed comparable performance to the commercially available preservation kit (R2 of 0.98), and samples consistently clustered by subject across each method. These data support that the DESS wipe method can be used for stable, room temperature collection and transport of human stool specimens.

Transcriptomic Network Interactions in Human Skin Treated with Topical Glucocorticoid Clobetasol Propionate.

Glucocorticoids are the most frequently used anti-inflammatory drugs in dermatology. However, the molecular signature of glucocorticoids and their receptor in human skin is largely unknown. Our validated bioinformatics analysis of human skin transcriptome induced by topical glucocorticoid clobetasol propionate (CBP) in healthy volunteers identified numerous unreported glucocorticoid-responsive genes, including over a thousand noncoding RNAs. We observed sexual and racial dimorphism in the CBP response including a shift toward IFN-α/IFN-γ and IL-6/Jak/Signal transducer and activator of transcription (STAT) 3 signaling in female skin; and a larger response to CBP in African-American skin. Weighted gene coexpression network analysis unveiled a dense skin network of 41 transcription factors including circadian Kruppel-like factor 9 (KLF9), and ∼260 of their target genes enriched for functional pathways representative of the entire CBP transcriptome. Using keratinocytes with Kruppel-like factor 9 knockdown, we revealed a feedforward loop in glucocorticoid receptor signaling, previously unreported. Interestingly, many of the CBP-regulated transcription factors were involved in the control of development, metabolism, circadian clock; and 80% of them were associated with skin aging showing similarities between glucocorticoid-treated and aged skin. Overall, these findings indicate that glucocorticoid receptor acts as an important regulator of gene expression in skin-both at the transcriptional and posttranscriptional level-via multiple mechanisms including regulation of noncoding RNAs and multiple core transcription factors.

Transcriptome Analysis Reveals Distinct Responses to Physiologic versus Toxic Manganese Exposure in Human Neuroblastoma Cells.

Manganese (Mn) is an essential trace element, which also causes neurotoxicity in exposed occupational workers. Mn causes mitochondrial toxicity; however, little is known about transcriptional responses discriminated by physiological and toxicological levels of Mn. Identification of such mechanisms could provide means to evaluate risk of Mn toxicity and also potential avenues to protect against adverse effects. To study the Mn dose-response effects on transcription, analyzed by RNA-Seq, we used human SH-SY5Y neuroblastoma cells exposed for 5 h to Mn (0 to 100 µM), a time point where no immediate cell death occurred at any of the doses. Results showed widespread effects on abundance of protein-coding genes for metabolism of reactive oxygen species, energy sensing, glycolysis, and protein homeostasis including the unfolded protein response and transcriptional regulation. Exposure to a concentration (10 µM Mn for 5 h) that did not result in cell death after 24-h increased abundance of differentially expressed genes (DEGs) in the protein secretion pathway that function in protein trafficking and cellular homeostasis. These include BET1 (Golgi vesicular membrane-trafficking protein), ADAM10 (ADAM metallopeptidase domain 10), and ARFGAP3 (ADP-ribosylation factor GTPase-activating protein 3). In contrast, 5-h exposure to 100 µM Mn, a concentration that caused cell death after 24 h, increased abundance of DEGs for components of the mitochondrial oxidative phosphorylation pathway. Integrated pathway analysis results showed that protein secretion gene set was associated with amino acid metabolites in response to 10 µM Mn, while oxidative phosphorylation gene set was associated with energy, lipid, and neurotransmitter metabolites at 100 µM Mn. These results show that differential effects of Mn occur at a concentration which does not cause subsequent cell death compared to a concentration that causes subsequent cell death. If these responses translate to effects on the secretory pathway and mitochondrial functions in vivo, differential activities of these systems could provide a sensitive basis to discriminate sub-toxic and toxic environmental and occupational Mn exposures.

Distinct amino acid and lipid perturbations characterize acute versus chronic malaria.

Chronic malaria is a major public health problem and significant challenge for disease eradication efforts. Despite its importance, the biological factors underpinning chronic malaria are not fully understood. Recent studies have shown that host metabolic state can influence malaria pathogenesis and transmission, but its role in chronicity is not known. Here, with the goal of identifying distinct modifications in the metabolite profiles of acute versus chronic malaria, metabolomics was performed on plasma from Plasmodium-infected humans and nonhuman primates with a range of parasitemias and clinical signs. In rhesus macaques infected with Plasmodium coatneyi, significant alterations in amines, carnitines, and lipids were detected during a high parasitemic acute phase and many of these reverted to baseline levels once a low parasitemic chronic phase was established. Plasmodium gene expression, studied in parallel in the macaques, revealed transcriptional changes in amine, fatty acid, lipid and energy metabolism genes, as well as variant antigen genes. Furthermore, a common set of amines, carnitines, and lipids distinguished acute from chronic malaria in plasma from human Plasmodium falciparum cases. In summary, distinct host-parasite metabolic environments have been uncovered that characterize acute versus chronic malaria, providing insights into the underlying host-parasite biology of malaria disease progression.

The persistence of parasitic plasmids.

The conditions under which plasmids are predicted to persist remain controversial. Here, we reevaluate the ordinary differential equations used previously to model plasmid persistence and conclude that the parameter space required for maintenance is far less stringent than has been supposed. Strikingly, our model demonstrates that purely parasitic plasmids may persist, even in the absence of heterogeneity in the host population, and that this persistence is expressed by oscillations or damped oscillations between the plasmid-bearing and the plasmid-free class.

Time-course analysis of microRNA-induced mesenchymal-to-epithelial transition underscores the complexity of the underlying molecular processes.

Expression levels of the miR-200 family of miRNAs are significantly reduced during the epithelial-to-mesenchymal transition (EMT) and consequent metastasis of ovarian and other cancers. Consistently, ectopic over-expression of miR-200 family miRNAs in mesenchymal-like cells reverses the process by converting treated cells to an epithelial phenotype, thereby reducing invasiveness and increasing sensitivity to chemotherapeutic drugs. To better understand the dynamics and molecular processes underlying miRNA-induced mesenchymal-to mesenchymal transition (MET), a time-course study was conducted where miRNA-induced morphological and molecular changes associated with MET were monitored over a period of 144 h. Morphological transition from an elongated mesenchymal-like to a cuboidal epithelial-like phenotype is maximized at 48 h with cells returning to the elongated phenotype by 144 h. Changes in the expression of >3000 genes, including many previously associated with epithelial-to-mesenchymal transition (EMT), are most pronounced at 48 h, and approach starting levels of expression by 144 h. The majority of these genes are not direct targets of miR-429. Targeted (siRNA) inhibition of key miR-429 regulated genes previously implicated as drivers of EMT/MET, do not recapitulate miR-429 induced MET indicating that the underlying molecular processes are complex.

Low-level maternal exposure to nicotine associates with significant metabolic perturbations in second-trimester amniotic fluid.

Decades of public health research have documented that smoking in pregnancy poses significant health risks to both mother and child. More recent studies have shown that even passive maternal exposure to secondhand smoke associates with negative birth outcomes. However, the mechanisms linking exposure to outcomes have remained obscure. As a first step toward defining the metabolic consequence of low-level nicotine exposure on fetal development, we conducted an untargeted metabolomic analysis of 81 paired samples of maternal serum and amniotic fluid collected from karyotypically normal pregnancies in the second trimester. By comparing the m/z and retention times of our mass spectral features with confirmed standards, we identified cotinine, a nicotine derivative, and used the calculated cotinine concentrations to classify our maternal serum samples into exposure groups using previously defined cut-offs. We found that cotinine levels consistent with low-level maternal exposure to nicotine associated with distinct metabolic perturbations, particularly in amniotic fluid. In fact, the metabolic effects in amniotic fluid of ostensibly low-level exposed mothers showed greater overlap with perturbations previously observed in the sera of adult smokers than did the perturbations observed in the corresponding maternal sera. Dysregulated fetal pathways included aspartate and asparagine metabolism, pyrimidine metabolism, and metabolism of other amino acids. We also observed a strong negative association between level of maternal serum cotinine and acetylated polyamines in the amniotic fluid. Combined, these results confirm that low-level maternal nicotine exposure, indicated by a maternal serum cotinine level of 2-10ng/mL, is associated with striking metabolic consequences in the fetal compartment, and that the affected pathways overlap those perturbed in the sera of adult smokers.

Claudin-based barrier differentiation in the colonic epithelial crypt niche involves Hopx/Klf4 and Tcf7l2/Hnf4-α cascades.

Colonic enterocytes form a rapidly renewing epithelium and barrier to luminal antigens. During renewal, coordinated expression of the claudin family of genes is vital to maintain the epithelial barrier. Disruption of this process contributes to barrier compromise and mucosal inflammatory diseases. However, little is known about the regulation of this critical aspect of epithelial cell differentiation. In order to identify claudin regulatory factors we utilized high-throughput gene microarrays and correlation analyses. We identified complex expression gradients for the transcription factors Hopx, Hnf4a, Klf4 and Tcf7l2, as well as 12 claudins, during differentiation. In vitro confirmatory methods identified 2 pathways that stimulate claudin expression; Hopx/Klf4 activation of Cldn4, 7 and 15, and Tcf7l2/Hnf4a up-regulation of Cldn23. Chromatin immunoprecipitation confirmed a Tcf7l2/Hnf4a/Claudin23 cascade. Furthermore, Hnf4a conditional knockout mice fail to induce Cldn23 during colonocyte differentiation. In conclusion, we report a comprehensive screen of colonic claudin gene expression and discover spatiotemporal Hopx/Klf4 and Tcf7l2/Hnf4a signaling as stimulators of colonic epithelial barrier differentiation.

Evidence for the importance of personalized molecular profiling in pancreatic cancer.

OBJECTIVES: There is a growing body of evidence that targeted gene therapy holds great promise for the future treatment of cancer. A crucial step in this therapy is the accurate identification of appropriate candidate genes/pathways for targeted treatment. One approach is to identify variant genes/pathways that are significantly enriched in groups of afflicted individuals relative to control subjects. However, if there are multiple molecular pathways to the same cancer, the molecular determinants of the disease may be heterogeneous among individuals and possibly go undetected by group analyses. METHODS: In an effort to explore this question in pancreatic cancer, we compared the most significantly differentially expressed genes/pathways between cancer and control patient samples as determined by group versus personalized analyses. RESULTS: We found little to no overlap between genes/pathways identified by gene expression profiling using group analyses relative to those identified by personalized analyses. CONCLUSIONS: Our results indicate that personalized and not group molecular profiling is the most appropriate approach for the identification of putative candidates for targeted gene therapy of pancreatic and perhaps other cancers with heterogeneous molecular etiology.

Molecular profiling predicts the existence of two functionally distinct classes of ovarian cancer stroma.

Although stromal cell signaling has been shown to play a significant role in the progression of many cancers, relatively little is known about its importance in modulating ovarian cancer development. The purpose of this study was to investigate the process of stroma activation in human ovarian cancer by molecular analysis of matched sets of cancer and surrounding stroma tissues. RNA microarray profiling of 45 tissue samples was carried out using the Affymetrix (U133 Plus 2.0) gene expression platform. Laser capture microdissection (LCM) was employed to isolate cancer cells from the tumors of ovarian cancer patients (Cepi) and matched sets of surrounding cancer stroma (CS). For controls, ovarian surface epithelial cells (OSE) were isolated from the normal (noncancerous) ovaries and normal stroma (NS). Hierarchical clustering of the microarray data resulted in clear separations between the OSE, Cepi, NS, and CS samples. Expression patterns of genes encoding signaling molecules and compatible receptors in the CS and Cepi samples indicate the existence of two subgroups of cancer stroma (CS) with different propensities to support tumor growth. Our results indicate that functionally significant variability exists among ovarian cancer patients in the ability of the microenvironment to modulate cancer development.

Molecular profiling supports the role of epithelial-to-mesenchymal transition (EMT) in ovarian cancer metastasis.

BACKGROUND: While metastasis ranks among the most lethal of all cancer-associated processes, on the molecular level, it remains one of the least well understood. One model that has gained credibility in recent years is that metastasizing cells at least partially recapitulate the developmental process of epithelial-to-mesenchymal transition (EMT) in their transit from primary to metastatic sites. While experimentally supported by cell culture and animal model studies, the lack of unambiguous confirmatory evidence in cancer patients has led to persistent challenges to the model's relevance in humans. METHODS: Gene expression profiling (Affymetrix, U133) was carried out on 14 matched sets of primary (ovary) and metastatic (omentum) ovarian cancer (serous adenocarcinoma) patient samples. Hierarchical clustering and functional pathway algorithms were used in the data analysis. RESULTS: While histological examination reveled no morphological distinction between the matched sets of primary and metastatic samples, gene expression profiling clearly distinguished two classes of metastatic samples. One class displayed expression patterns statistically indistinguishable from primary samples isolated from the same patients while a second class displayed expression patterns significantly different from primary samples. Further analyses focusing on genes previously associated with EMT clearly distinguished the primary from metastatic samples in all but one patient. CONCLUSION: Our results are consistent with a role of EMT in most if not all ovarian cancer metastases and demonstrate that identical morphologies between primary and metastatic cancer samples is insufficient evidence to negate a role of EMT in the metastatic process.