Potential Role of Extracellular ATP Released by Bacteria in Bladder Infection and Contractility.

Urgency urinary incontinence (UUI) and overactive bladder (OAB) can both potentially be influenced by commensal and urinary tract infection-associated bacteria. The sensing of bladder filling involves interplay between various components of the nervous system, eventually resulting in contraction of the detrusor muscle during micturition. This study models host responses to various urogenital bacteria, first by using urothelial bladder cell lines and then with myofibroblast contraction assays. To measure responses, we examined Ca2+ influx, gene expression, and alpha smooth muscle actin deposition assays. Organisms such as Escherichia coli and Gardnerella vaginalis were found to strongly induce Ca2+ influx and contraction, whereas Lactobacillus crispatus and L. gasseri did not induce this response. Additionally, supernatants from lactobacilli impeded Ca2+ influx and contraction induced by uropathogens. Upon further investigation of factors associated with purinergic signaling pathways, the Ca2+ influx and contraction of cells correlated with the amount of extracellular ATP produced by E. coli Certain lactobacilli appear to mitigate this response by utilizing extracellular ATP or producing inhibitory compounds that may act as a receptor agonist or Ca2+ channel blocker. These findings suggest that members of the urinary microbiota may be influencing UUI or OAB.IMPORTANCE The ability of uropathogenic bacteria to release excitatory compounds, such as ATP, may act as a virulence factor to stimulate signaling pathways that could have profound effects on the urothelium, perhaps extending to the vagina. This may be countered by the ability of certain commensal urinary microbiota constituents, such as lactobacilli. Further understanding of these interactions is important for the treatment and prevention of UUI and OAB. The clinical implications may require a more targeted approach to enhance the commensal bacteria and reduce ATP release by pathogens.

Development of a continuous cell line from larval Atlantic cod (Gadus morhua) and its use in the study of the microsporidian, Loma morhua.

In vitro cell culture methods are crucial for the isolation, purification and mass propagation of intracellular pathogens of aquatic organisms. Cell culture infection models can yield insights into infection mechanisms, aid in developing methods for disease mitigation and prevention, and inform commercial-scale cultivation approaches. This study details the establishment of a larval cell line (GML-5) from the Atlantic cod (Gadus morhua) and its use in the study of microsporidia. GML-5 has survived over 100 passages in 8 years of culture. The line remains active and viable between 8 and 21°C in Leibovitz-15 (L-15) media with 10% foetal bovine serum and exhibits a myofibroblast phenotype as indicated by immuno-positive results for vimentin, α-smooth muscle actin, collagen I and S-100 proteins, while being desmin-negative. GML-5 supports the infection and development of two microsporidian parasites, an opportunistic generalist (Anncaliia algerae) and cod-specific Loma morhua. Using GML-5, spore germination and proliferation of L. morhua was found to require exposure to basic pH and cool incubation temperatures (8°C), in contrast to A. algerae, which required no cultural modifications. Loma morhua-associated xenoma-like structures were observed 2 weeks postexposure. This in vitro infection model may serve as a valuable tool for cod parasitology and aquaculture research.

Lactobacillus rhamnosus GG increases cyclooxygenase-2 expression and prostaglandin E2 secretion in colonic myofibroblasts via a MyD88-dependent mechanism during homeostasis.

Prostaglandin E2 (PGE2 ) plays a critical role in intestinal mucosal tolerance and barrier integrity. Cyclooxygenase-2 (COX-2)-dependent PGE2 production involves mobilisation of arachidonic acid. Lactobacillus rhamnosus GG (LbGG) is one of the most widely used probiotics reported to colonise the colonic mucosa. LbGG contributes to the protection of the small intestine against radiation injury through the repositioning of mucosal COX-2 expressing cells. However, it is unknown if LbGG modulates PGE2 production in the colonic mucosa under homeostasis and the major cellular elements involved in these processes. Colonic epithelial and CD90+ mesenchymal stromal cells, also known as (myo) fibroblasts (CMFs), are abundant innate immune cells in normal colonic mucosa able to produce PGE2 . Herein, we tested the hypothesis that under colonic mucosal homeostasis, LbGG modulates the eicosanoid pathway resulting in increased PGE2 production in both epithelial and stromal cells. Among the five tested human colonic epithelial cell lines, only exposure of Caco-2 to LbGG for 24 hr led to the mobilisation of arachidonic acid with concomitant increase in the components within the leukotriene and COX-2-dependent PGE2 pathways. By contrast, CMFs isolated from the normal human colonic mucosa responded to LbGG with increased expression of COX-2 and PGE2 in the prostaglandin pathway, but not 5-LO in the leukotriene pathway. Oral gavage of C57BL/6 mice for 5 days with LbGG (5 × 108 Colony-Forming Unit (CFU)/dose) increased COX-2 expression in the colonic mucosa. The majority of cells upregulating COX-2 protein expression were located in the colonic lamina propria and colocalised with α-SMA+ cells corresponding to the CMF phenotype. This process was myeloid differentiation factor-88-dependent, because silencing of myeloid differentiation factor-88 expression in CMFs abrogated LbGG-induced upregulation of COX-2 in culture and in vivo. Taken together, our data suggest that LbGG increases release of COX-2-mediated PGE2 , contributing to the maintenance of mucosal homeostasis in the colon and CMFs are among the major contributors to this process.

Molecular Pathogenesis of Chlamydia Disease Complications: Epithelial-Mesenchymal Transition and Fibrosis.

The reproductive system complications of genital chlamydial infection include fallopian tube fibrosis and tubal factor infertility. However, the molecular pathogenesis of these complications remains poorly understood. The induction of pathogenic epithelial-mesenchymal transition (EMT) through microRNA (miRNA) dysregulation was recently proposed as the pathogenic basis of chlamydial complications. Focusing on fibrogenesis, we investigated the hypothesis that chlamydia-induced fibrosis is caused by EMT-driven generation of myofibroblasts, the effector cells of fibrosis that produce excessive extracellular matrix (ECM) proteins. The results revealed that the targets of a major category of altered miRNAs during chlamydial infection are key components of the pathophysiological process of fibrogenesis; these target molecules include collagen types I, III, and IV, transforming growth factor ß (TGF-ß), TGF-ß receptor 1 (TGF-ßR1), connective tissue growth factor (CTGF), E-cadherin, SRY-box 7 (SOX7), and NFAT (nuclear factor of activated T cells) kinase dual-specificity tyrosine (Y) phosphorylation-regulated kinase 1a (Dyrk1a). Chlamydial induction of EMT resulted in the generation of α-smooth muscle actin (α-SMA)-positive myofibroblasts that produced ECM proteins, including collagen types I and III and fibronectin. Furthermore, the inhibition of EMT prevented the generation of myofibroblasts and production of ECM proteins during chlamydial infection. These findings may provide useful avenues for targeting EMT or specific components of the EMT pathways as a therapeutic intervention strategy to prevent chlamydia-related complications.

Primary mouse lung fibroblasts help macrophages to tackle Mycobacterium tuberculosis more efficiently and differentiate into myofibroblasts up on bacterial stimulation.

Keeping with their classical role in wound healing, fibroblasts of the lung take part in the resolution of tubercular granulomas. They are totally absent in nascent granulomas, but surround necrotizing granulomas, and are the majority of cells in healed granulomas. Lung fibroblasts may become infected with Mycobacterium tuberculosis (Mtb). Two previous studies suggested an immunomodulatory effect of fibroblasts on infected macrophages. In the present study, we looked at the role of primary mouse lung fibroblasts on naive or activated mouse bone marrow macrophages infected with Mtb and the effect of infection on fibroblast properties. We observed that with fibroblasts in the vicinity, infected naive macrophages restricted the bacterial growth, while activated macrophages turned more bactericidal with concomitant increase in nitrite production. Neutralizing IL-1α in fibroblast supernatant reduced the nitrite production by infected macrophages. Secretion of IL-6 and MCP-1 was down-regulated, while TNF-α was up-regulated in infected naive macrophages. In infected activated macrophages, the secretion of IL-6 was up-regulated, while that of MCP-1 and TNF-α was unaffected. The 'fibroblast effects' were enhanced when the fibroblasts too were infected. Mtb induced IL-1 secretion and pro-fibrotic responses by fibroblasts. Mtb-induced myofibroblast conversion was blocked by rapamycin suggesting cell signalling via mTOR.

Molecular alterations in fibroblasts exposed to Helicobacter pylori: a missing link in bacterial inflammation progressing into gastric carcinogenesis?

Major human pathogen Helicobacter pylori (Hp) can colonize the gastric mucosa causing inflammation and being of potential risk for gastric cancer development but the contribution of fibroblasts to the pathogenesis of Hp in the stomach has been little studied. Normal stroma contains few fibroblasts, especially myofibroblasts, but their number rapidly increases in the reactive stroma surrounding inflammatory region and neoplastic tissue. We determined the effect of coincubation of cultured rat gastric fibroblasts with alive Hp on the transdifferentiation of fibroblasts into myofibroblasts associated with Hp-induced inflammation and neoplasia. Gastric mucosal samples were harvested from 8-week-old Spraque-Dowley rats and cultured to obtain the sub-confluent fibroblasts. The isolated fibroblasts were infected with 1 x 10(9) of live Hp (ATCC 700824, cagA+, vacA+) per dish and incubated in humidified atmosphere for 3, 24 and 48 hours. At respective times, fibroblasts were harvested and the expression of mRNA for α-smooth muscle actin (SMA), hypoxia inducible factor (HIF)-1α, collagen I, heat shock protein (HSP)-70, heme oxygenase (HO)-1, Bax and Ki67 transcripts was determined by RT-PCR with specific primers. Hp increased the transdifferentiation of fibroblasts into myofibroblasts as reflected by the time-dependent overexpression of mRNA for α-SMA. The increased expression of HIF-1α and collagen I was observed in fibroblasts co-cultured with Hp. The expression of HSP70 which was negligible in isolated fibroblasts incubated with vehicle (saline) showed time-dependent 2-3 fold increase in those incubated with Hp. The HO-1 mRNA was strongly expressed in rat gastric fibroblasts without or with the co-incubation with Hp. The mRNA for Bax was progressively downregulated within the time of incubation while no significant changes in expression of proliferation marker Ki67 were recorded. We conclude that Hp-induced transdifferentiation of fibroblasts into myofibroblasts involves an increased expression of the early carcinogenic marker HIF-1α, and inhibition of proapoptotic Bax expression, and 2) the overexpression of HSP70 and the unchanged expression HO-1 and Ki67 probably represent the enhanced protective activity of Hp-infected fibroblasts to maintain their own integrity under inflammatory action of this bacteria and its cytotoxins.

Stromal cells induce Th17 during Helicobacter pylori infection and in the gastric tumor microenvironment.

Gastric cancer is associated with chronic inflammation and Helicobacter pylori infection. Th17 cells are CD4(+) T cells associated with infections and inflammation; but their role and mechanism of induction during carcinogenesis is not understood. Gastric myofibroblasts/fibroblasts (GMF) are abundant class II MHC expressing cells that act as novel antigen presenting cells. Here we have demonstrated the accumulation of Th17 in H. pylori-infected human tissues and in the gastric tumor microenvironment. GMF isolated from human gastric cancer and H. pylori infected tissues co-cultured with CD4(+) T cells induced substantially higher levels of Th17 than GMF from normal tissues in an IL-6, TGF-ß, and IL-21 dependent manner. Th17 required interaction with class II MHC on GMF for activation and proliferation. These studies suggest that Th17 are induced during both H. pylori infection and gastric cancer in the inflammatory milieu of gastric stroma and may be an important link between inflammation and carcinogenesis.

Folic acid increases global DNA methylation and reduces inflammation to prevent Helicobacter-associated gastric cancer in mice.

BACKGROUND & AIMS: Previous studies have suggested that dietary folic acid (FA) can protect against certain types of cancers. However, the findings have varied, and the mechanisms by which FA exerts chemopreventive effects remain to be clarified. We examined the effects of FA supplementation on DNA methylation, gene expression, and gastric dysplasia in a transgenic mouse model that is etiologically and histologically well matched with human gastric cancers. METHODS: Hypergastrinemic mice infected with Helicobacter felis were studied at multiple stages of gastric dysplasia and early cancer with FA supplementation initiated both at weaning and later in life. Global DNA methylation was assessed by a methylation sensitive cytosine incorporation assay, bisulfite pyrosequencing of B1 repetitive elements, and immunohistochemistry with anti-5-methylcytosine. We also profiled gene expression in the same tissues. RESULTS: We found a decrease in global DNA methylation and tissue folate and an increase in serum homocysteine with progression of gastric dysplasia. FA supplementation prevented this loss of global DNA methylation and markedly reduced gastric dysplasia and mucosal inflammation. FA protected against the loss of global DNA methylation both in the dysplastic gastric epithelial cells and in gastric stromal myofibroblasts. In addition, FA supplementation had an anti-inflammatory effect, as indicated by expression profiling and immunohistochemistry for lymphocyte markers. CONCLUSIONS: We conclude that FA supplementation is chemopreventive in this model of Helicobacter-associated gastric cancer. The beneficial effect of FA is likely due to its ability to prevent global loss of methylation and suppress inflammation.

The thermolysin-like metalloproteinase and virulence factor LasB from pathogenic Pseudomonas aeruginosa induces anoikis of human vascular cells.

Disruption of cell/ECM interactions resulting from uncontrolled pericellular proteolysis leads to detachment-induced cell apoptosis (anoikis), contributing to the morbid evolution of inflammatory vascular diseases. During cardiovascular infections, bacterial proteinases might induce vascular cells to enter a similar pathway. We focused on LasB, the predominant metalloproteinase secreted by the haematotropic pathogen Pseudomonas aeruginosa. While the exosecretome of the LasB-deficient pseudomonal strain PAO1lasBΔ had limited impact on human vascular cell adherence and viability, secretomes from the LasB-expressing reference strain, PAO1, or clinical isolates from patients with cardiac infection all induced anoikis, as did purified LasB. Immunofluorescence and/or immunoblotting analysis of heart valve myofibroblast cultures or whole tissue revealed an extensive, LasB-dependent degradation of ECM-associated fibronectin and vitronectin, that preceded cell de-adherence, whereas type I collagen showed limited degradation. Moreover, LasB produced a rapid endoproteolysis of the cell-associated urokinase receptor/uPAR, leaving a truncated receptor that is unable to support cell adherence and survival via interactions with vitronectin and integrins. Conversely, major myofibroblast integrins showed no or only minor alterations. Thus, among P. aeruginosa-secreted metalloproteinases, LasB can induce vascular cell anoikis through simultaneous proteolysis of ECM components and cell receptors, suggesting the uPAR-vitronectin axis as a major target in this process.