The danger signal adenosine induces persistence of chlamydial infection through stimulation of A2b receptors

Infections with intracellular bacteria such as chlamydiae affect the majority of the world population. Infected tissue inflammation and granuloma formation help contain the short-term expansion of the invading pathogen, leading also to local tissue damage and hypoxia. However, the effects of key aspects of damaged inflamed tissues and hypoxia on continued infection with intracellular bacteria remain unknown. We find that development of Chlamydia trachomatis is reversibly retarded by prolonged exposure of infected cells to extracellular adenosine, a hallmark of hypoxia and advanced inflammation. In epithelial cells, this effect was mediated by the A2b adenosine receptor, unique in the adenosine receptor family for having a hypoxia-inducible factor (HIF1-α) binding site at its promoter region, and was dependent on an increase in the intracellular cAMP levels, but was independent of cAMP-dependent protein kinase (PKA). Further study of adenosine receptor signaling during intracellular bacterial infection could lead to breakthroughs in our understanding of persistent infections with these ubiquitous pathogens

The Danger Signal Adenosine Induces Persistence of Chlamydial Infection through Stimulation of A2b Receptors

Infections with intracellular bacteria such as chlamydiae affect the majority of the world population. Infected tissue inflammation and granuloma formation help contain the short-term expansion of the invading pathogen, leading also to local tissue damage and hypoxia. However, the effects of key aspects of damaged inflamed tissues and hypoxia on continued infection with intracellular bacteria remain unknown. We find that development of Chlamydia trachomatis is reversibly retarded by prolonged exposure of infected cells to extracellular adenosine, a hallmark of hypoxia and advanced inflammation. In epithelial cells, this effect was mediated by the A2b adenosine receptor, unique in the adenosine receptor family for having a hypoxia-inducible factor (HIF1-α) binding site at its promoter region, and was dependent on an increase in the intracellular cAMP levels, but was independent of cAMP-dependent protein kinase (PKA). Further study of adenosine receptor signaling during intracellular bacterial infection could lead to breakthroughs in our understanding of persistent infections with these ubiquitous pathogens

Ivermectin Inhibits Growth of Chlamydia trachomatis in Epithelial Cells

Ivermectin is currently approved for treatment of both clinical and veterinary infections by nematodes, including Onchocerca cervicalis in horses and Onchocerca volvulus in humans. However, ivermectin has never been shown to be effective against bacterial pathogens. Here we show that ivermectin also inhibits infection of epithelial cells by the bacterial pathogen, Chlamydia trachomatis, at doses that could be envisioned clinically for sexually-transmitted or ocular infections by Chlamydia

Reversible inhibition of Chlamydia trachomatis infection in epithelial cells due to stimulation of P2X4 receptors

Bacterial infections of the mucosal epithelium are a major cause of human disease. The prolonged presence of microbial pathogens stimulates inflammation of the local tissues, which leads to changes in the molecular composition of the extracellular milieu. A well-characterized molecule that is released to the extracellular milieu by stressed or infected cells is extracellular ATP and its ecto-enzymatic degradation products, which function as signaling molecules through ligation of purinergic receptors. There has been little information, however, on the effects of the extracellular metabolites on bacterial growth in inflamed tissues. Millimolar concentrations of ATP have been previously shown to inhibit irreversibly bacterial infection through ligation of P2X7 receptors. We show here that the proinflammatory mediator, ATP, is released from Chlamydia trachomatis-infected epithelial cells. Moreover, further stimulation of the infected cells with micromolar extracellular ADP or ATP significantly impairs the growth of the bacteria, with a profile characteristic of the involvement of P2X4 receptors. A specific role for P2X4 was confirmed using cells overexpressing P2X4. The chlamydiae remain viable and return to normal growth kinetics after removal of the extracellular stimulus, similar to responses previously described for persistence of chlamydial infection

Ivermectin is not cytotoxic to epithelial cells.

<p>(A) HeLa cells were cultured in the presence of the indicated concentrations of ivermectin or vehicle (0.2% ethanol), supernatants were then centrifuged at 500 g for 5 minutes, and adherent cells were removed with trypsin/EDTA and gently recombined with supernatant pellets. Cell viability was determined by trypan blue exclusion, evaluated using a hemocytometer. (B) Supernatants from the cultures described in panel A, and from wells treated with 2% Triton X-100 30 minutes prior to collection time points, were combined and evaluated for LDH activity using a Roche Cytotoxicity Detection Kit per the manufacturer’s instructions. Shown are means plus standard deviations for 3 independent experiments.</p

Ivermectin inhibits chlamydial infection of epithelial cells.

<p>(A) HeLa cells were infected with <i>C. trachomatis</i> serovar L2 at an MOI of 1, followed by treatment with ivermectin at the indicated concentrations at 1 hour post infection (hpi). Samples were harvested at 24 hpi for quantification of reinfectious yield (IFU/ml) on new HeLa cell monolayers utilizing fluorescent microscopy and anti-<i>C. trachomatis</i> antibodies. The values show means plus standard deviation of three independent experiments. (n = 3,**, P = 0.0059). (B) Total RNA was harvested at 24 hpi for quantification of chlamydial 16s rRNA. The values shown are relative to control values for each experiment, and are means and standard deviations of 3 independent experiments (n = 3, **, P = 0.0034, ***, P<0.001, compared to 100 nM condition). Two-tailed unpaired t tests were performed using GraphPad Prism version 5.0b for Mac.</p

Ivermectin inhibits the development of chlamydial inclusions in epithelial cells.

<p>HeLa cells grown on glass coverslips were infected with C. trachomatis serovar L2 at an MOI of 1, followed by treatment with ivermectin at the indicated concentrations at 1 hour post infection (hpi). At 24 hpi, cells were fixed with ice cold methanol for 10 minutes, followed by staining with <i>C. trachomatis</i> genus antibodies (Argene) and Hoechst (Sigma), and observed on a widefield fluorescence microscope (Leica).</p

Reversible inhibition of Chlamydia trachomatis infection in epithelial cells due to stimulation of P2X4 receptors

Bacterial infections of the mucosal epithelium are a major cause of human disease. The prolonged presence of microbial pathogens stimulates inflammation of the local tissues, which leads to changes in the molecular composition of the extracellular milieu. A well-characterized molecule that is released to the extracellular milieu by stressed or infected cells is extracellular ATP and its ecto-enzymatic degradation products, which function as signaling molecules through ligation of purinergic receptors. There has been little information, however, on the effects of the extracellular metabolites on bacterial growth in inflamed tissues. Millimolar concentrations of ATP have been previously shown to inhibit irreversibly bacterial infection through ligation of P2X7 receptors. We show here that the proinflammatory mediator, ATP, is released from Chlamydia trachomatis-infected epithelial cells. Moreover, further stimulation of the infected cells with micromolar extracellular ADP or ATP significantly impairs the growth of the bacteria, with a profile characteristic of the involvement of P2X4 receptors. A specific role for P2X4 was confirmed using cells overexpressing P2X4. The chlamydiae remain viable and return to normal growth kinetics after removal of the extracellular stimulus, similar to responses previously described for persistence of chlamydial infection

Reversible Inhibition of Chlamydia trachomatis Infection in Epithelial Cells Due to Stimulation of P2X 4

Bacterial infections of the mucosal epithelium are a major cause of human disease. The prolonged presence of microbial pathogens stimulates inflammation of the local tissues, which leads to changes in the molecular composition of the extracellular milieu. A well-characterized molecule that is released to the extracellular milieu by stressed or infected cells is extracellular ATP and its ecto-enzymatic degradation products, which function as signaling molecules through ligation of purinergic receptors. There has been little information, however, on the effects of the extracellular metabolites on bacterial growth in inflamed tissues. Millimolar concentrations of ATP have been previously shown to inhibit irreversibly bacterial infection through ligation of P2X(7) receptors. We show here that the proinflammatory mediator, ATP, is released from Chlamydia trachomatis-infected epithelial cells. Moreover, further stimulation of the infected cells with micromolar extracellular ADP or ATP significantly impairs the growth of the bacteria, with a profile characteristic of the involvement of P2X(4) receptors. A specific role for P2X(4) was confirmed using cells overexpressing P2X(4). The chlamydiae remain viable and return to normal growth kinetics after removal of the extracellular stimulus, similar to responses previously described for persistence of chlamydial infection