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

Abstract

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-alpha) 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.

Figure 1. Extended exposure to extracellular adenosine (ADO) causes a dose-dependent reduction in chlamydial development via the A2b adenosine receptor.

(A) HeLa cells were infected with C. trachomatis serovar L2 at an MOI of 1, followed by treatment with the adenosine deaminase inhibitor, EHNA, and ADO at indicated concentrations at 1 and 20 hours post infection (hpi). Samples were harvested at 42 hpi for quantification of reinfectious yield (IFU/ml) on new cell monolayers. The values show averages and S.D. from 3 samples of a representative experiment, and give results obtained from three independent experiments. (B) HeLa cell knock-downs (KDs) were produced with non-targeted (Control), A2a-targeted (A2a KD) or A2b-targeted (A2b KD) shRNA and levels of target KD verified to be decreased at least 50% by qPCR. Cells were infected and harvested as (A), with treatment of 25 µM EHNA and 50 µM ADO at 1 and 20 hpi. The values shown are averages and S.D. of values from 4 independent experiments. (n = 4, *, P<0.05, for treated cells in A2b KD compared to nontarget control.)

Figure 2. Repeated exposure to cell permeable cAMP analog or cAMP elevating agents causes a reduction in chlamydial development.

HeLa cells were infected with C. trachomatis serovar L2 at an MOI of 1, followed by treatment with adenosine (100 µM), forskolin (FSK, 10 µM), or 8-Br-cAMP (100 µM) starting at 1 hour post infection (hpi) every half-hour through 6 hpi (10 additions). Total RNA was harvested at 24 hpi for quantification of chlamydial 16s rRNA production using qPCR as indicated in methods. The values shown are relative to control values for each experiment, and are averages and S.D. from 3 independent experiments. (n = 3, **, P<0.0005, ***, P<0.0001 for treated cells compared to untreated control.)

Figure 3. Effects of adenosine on C. trachomatis growth are independent of PKA.

(A) HeLa cells were transfected PKA C-α targeted siRNA, then infected 30 hours post transfection with C. trachomatis, and treated with EHNA (25 µM) and adenosine (50 µM) 1 hpi. Total RNA was harvested at 24 hpi for quantification of chlamydial 16s rRNA production using qPCR as indicated in Materials and Methods. The values shown are relative to control values for each experiment, and are averages and S.D. from 3 independent experiments. (B) Western blot confirmation of PKA C-α depletion by PKA siRNA but not nontarget siRNA. Western blot was performed twice on two separate cell samples.

Figure 4. Exposure to adenosine induces a reversible block in chlamydial development.

HeLa cells were infected with C. trachomatis serovar L2 at an MOI of 1, followed by treatment with an equivalent volume of vehicle alone (control) or 25 µM EHNA and 50 µM adenosine at 1 hpi (non-control samples), 24 hpi (48hpi recovery; and Continuous Ado), and 48 hpi (Continuous Ado only). Media was refreshed in all samples at 24 and 48 hpi, and EHNA and adenosine added again to the samples indicated. Total RNA was harvested at 24 hpi for quantification of chlamydial 16s rRNA production using qPCR as indicated in methods. The values shown are averages plus S.D. of 3 independent experiments.

Figure 5. Extended exposure to adenosine causes modification of Chlamydia inclusions, tending to moderately sized inclusions with few chlamydiae, compared to smaller-sized inclusions with larger chlamydial forms following treatment with IFN-γ.

HeLa cells were infected with C. trachomatis serovar L2 at an MOI of 1.0. Samples were (A, D) untreated, (B, E) treated with 25 µM EHNA and 50 µM ADO at 1 and 20 hpi, or (C) treated with 5 ng/ml IFN-γ at 1 hpi. Staining with Acridine Orange was performed as described in Methods with fixation at 42 hpi. Red staining corresponds to RNA localization, while green staining corresponds to DNA. Arrows, Chlamydia inclusion; arrowheads, host-cell nuclei. Representative of three experiments, scale bars 10 µm. Electron microscopy samples were also fixed at 42 hpi and prepared for transmission electron microscopy as described in supporting material. Scale bars, 5 µm.