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Adenosine A2A receptor activation reduces recurrence and mortality from Clostridium difficileinfection in mice following vancomycin treatment
© Li et al.; licensee BioMed Central Ltd. 2012
Received: 23 July 2012
Accepted: 3 December 2012
Published: 10 December 2012
Activation of the A2A adenosine receptor (A2AAR) decreases production of inflammatory cytokines, prevents C. difficile toxin A-induced enteritis and, in combination with antibiotics, increases survival from sepsis in mice. We investigated whether A2AAR activation improves and A2AAR deletion worsens outcomes in a murine model of C. difficile (strain VPI10463) infection (CDI).
C57BL/6 mice were pretreated with an antibiotic cocktail prior to infection and then treated with vancomycin with or without an A2AAR agonist. A2AAR-/- and littermate wild-type (WT) mice were similarly infected, and IFNγ and TNFα were measured at peak of and recovery from infection.
Infected, untreated mice rapidly lost weight, developed diarrhea, and had mortality rates of 50-60%. Infected mice treated with vancomycin had less weight loss and diarrhea during antibiotic treatment but mortality increased to near 100% after discontinuation of antibiotics. Infected mice treated with both vancomycin and an A2AAR agonist, either ATL370 or ATL1222, had minimal weight loss and better long-term survival than mice treated with vancomycin alone. A2AAR KO mice were more susceptible than WT mice to death from CDI. Increases in cecal IFNγ and blood TNFα were pronounced in the absence of A2AARs.
In a murine model of CDI, vancomycin treatment resulted in reduced weight loss and diarrhea during acute infection, but high recurrence and late-onset death, with overall mortality being worse than untreated infected controls. The administration of vancomycin plus an A2AAR agonist reduced inflammation and improved survival rates, suggesting a possible benefit of A2AAR agonists in the management of CDI to prevent recurrent disease.
Clostridium difficile infection (CDI) is characterized by intense intestinal and systemic inflammatory reactions, especially in moderate to severe disease. Such microorganism-initiated tissue damage causes de novo production and in situ accumulation of adenosine that signals through four G protein–coupled receptors designated as A1, A2A, A2B, and A3. Activation of the A2A adenosine receptor (A2AAR) produces a constellation of responses that are anti-inflammatory. Pro-inflammatory responses in bone marrow derived cells (BMDC) including platelets , monocytes , mast cells [4, 5], neutrophils [6–8] and T cells [9–11] are all inhibited by A2AAR activation.
Adenosine is a purine nucleoside that plays an important role in many biochemical processes such as energy transfer. It also acts as a secondary messenger and neurotransmitter . Endogenous adenosine is produced in part by nucleotide degradation with participation of 5′ nucleotidase with or without ectonucleotidases on cell membranes, from the pool of adenosine triphosphate (ATP), adenosine diphosphate (ADP), or adenosine monophosphate (AMP) released through regulatory processes, inflammation, or cellular damage. Adenosine can further be degraded to inosine by adenosine deaminase intra- and/or extra-cellularly. Otherwise, it can be retaken up and converted back to AMP by adenosine kinase. As an endogenous purine nucleotide, adenosine modulates many physiological processes through four adenosine receptor subtypes, A1, A2A, A2B and A3.
Selective activation of the A2AAR with synthetic adenosine analogs has been demonstrated to protect many tissues, including liver, kidney, skin, heart, and spinal cord, from ischemia-reperfusion injury [13–16], to inhibit inflammatory responses in rabbit joint sepsis induced by LPS , and to improve mouse survival from sepsis with Escherichia coli [18, 19] or Staphylococcus aureus  in combination with antibiotic treatment. Previous studies have suggested that activation of A2AARs with ATL 313, or inhibition of adenosine deaminase prevents Clostridium difficile toxin A-induced enteritis by reducing the production of inflammatory cytokines in mouse or rabbit ileal loop model [20–22]. In the current study, we found that A2AAR activation during antibiotic treatment for CDI lessens disease severity, prevents relapse and increases survival of mice. Deletion of A2AARs worsens outcome of CDI by enhancing the host inflammatory response to infection. The beneficial effects of A2AR activation are probably caused by anti-inflammatory effects of A2AAR activation counteracting the pro-inflammatory effects of C. difficile toxins.
Eight-week old male C57BL/6 mice were purchased from the Jackson Laboratory (Bar Harbor, ME 04609). Food and water were provided ad libitum before and during the experiments. A2AAR -/- mice from Jiang-Fan Chen  of Boston University were bred to be congenic with C57BL/6 mice. A2AAR -/- mice were age- and sex-matched to wild type controls. Mouse genotyping employed a set of 3 primers (5′-GGGCTCCTCGGTGTACAT-3′, 5′-CCCACAGATCTAGCCTTA-3′, 5′-TGTCACGTCCTGCACGAC-3′) to resolve a 380-bp wild type allele versus a 500-bp knockout allele. Animals were housed in a pathogen-free isolation barrier facility with chip bedding. A previously published infection model was adapted with slight modification . Briefly, all mice were started with a 3-day antibiotic cocktail pretreatment containing 4.5 mg of vancomycin, 4.2 units of colistin, 3.5 mg of gentamicin, and 21.5 mg of metronidazole per kg/day in drinking water 6 days before the infection. Clindamycin (32 mg/kg) was given intraperitoneally to each mouse the day before the infection. Mice were transferred from a pathogen-free room to a BSL-2 room within the vivarium where they were prepared for infection. Infected mice remained in the same cage and were placed in a dedicated sash in the BSL-2 room. In most experiments, 50 mg/kg/day of vancomycin was administered in drinking water starting 24 hours post infection. The vancomycin treatment was routinely terminated on day 4 post infection unless specifically stated. Treatment with an A2AAR agonist (via Alzet pump as described below) was typically started 1 day post-infection (unless specified) and lasted for either 7 or 14 days. Mice that were considered moribund were euthanized by cervical dislocation. Infection and treatment protocol was approved by the University of Virginia Animal Care and Use Committee.
Radioligand binding assays
The adenosine receptor binding assay methodology has been described previously  and was conducted by Dogwood Pharmaceuticals (Charlottesville, VA 22911). In brief, all subtypes of recombinant human and mouse ARs were stably expressed in HEK-293 cells (A2AAR, A2BAR, and A3AR), CHO-K1 cells (A1AR) or transiently expressed by baculoviral infection of Sf9 cells (A2AAR, high-affinity assay). Crude membranes were prepared from these transfected cells. An appropriate radioligand (125I-ABA for A1AR and A3AR, 125I-ZM241385 for A2AAR, 125I-ABOPX for A2B AR, or 125I-APE for A2AAR/high affinity assay) was added, then filtered through glass fiber filters, and counted in a Wallac Wizard 1470 gamma counter (Perkin Elmer, Boston MA). The non-specific binding of radiolabeled ligand was measured in the presence of the non-selective AR agonist, NECA (100 μM). G protein coupled receptors bound agonists with two affinity states (G protein coupled and G protein uncoupled). The coupled high affinity state was the more relevant assay because it reflects the active configuration of the receptor. To produce G protein coupled human A2AARs, Sf9 cells were simultaneously infected with 4 baculoviruses encoding the human A2AAR and G protein αs, β4 and γ2 subunits . High affinity binding was performed using 125I-APE as described above. Competition binding curves were constructed and IC50 values calculated using a 4-parameter logistic fit (PRISM 5.0, GraphPad Software, San Diego, CA). The value of K i for competition for radioligand binding by agonist was calculated using the Cheng-Prusoff equation . K i values were calculated from the mean of triplicate or quadruplicate assays and normalized to the mean value of the inter-assay standard.
Preparation of Clostridium difficileinocula
C. difficile was grown for 20 hours in CMB (37°C), transferred to a fresh tube of CMB and incubated for 5 hours to achieve log-phase growth prior to infection. Bacteria were centrifuged at 10,000 rpm for 2 minutes, washed with BHI medium three times, reconstituted with BHI medium, and quantified by spectrophotometry. An OD reading of 1.0 was calculated to be equivalent to 5×107 cfu C. difficile/ml. The final concentration of C. difficile (cfu/ml) was validated by hemocytometer reading under light microscopy. Unless the dose was specifically stated, 1×104-5 of C. difficile was gavaged to each mouse in the infected groups, and BHI medium only was given to mice in uninfected groups.
Implantation of ALZET osmotic pumps
ALZET mini-osmotic pumps (Model 1007D and 1002) were implanted in mice using a protocol approved by the University of Virginia Animal Care and Use Committee. Briefly, mice were anesthetized with a mixture of ketamine and xylazine and placed on a heating pad. A short (~5 mm) incision was made at the interscapular area after shaving and cleaning the skin with 70% ethanol and iodine. Pumps filled with A2AAR agonists (ATL370 or ATL1222 at either 1 or 10 ng/kg/min) for treated mice or an equivalent amount of the vehicle-3% dimethyl sulfoxide (DMSO) in PBS only, for untreated control mice were inserted subcutaneously. A wound clip was used to close the incision. Mice were allowed to wake up on the heating pad before returning to the BSL2 sash.
Clinical scoring system
Clinical scoring system for mice infected with Clostridium difficile
Inactive unless prodded
Very ruffled/puff/ Ungroomed
Soft stool/discolored (yellowish)
Wet stained tail/ mucous +/- blood
Liquid/no stool (ileus)
Squinted ½ closed
Extraction of DNA from frozen mouse stools
All stool samples were weighed before DNA extraction for sample normalization. Stool DNA was extracted under the modified protocol provided in the QIAamp DNA Stool Mini Kit. Briefly, frozen stool was added to 400 μL of ASL buffer, homogenized by grinding with a wooden stick, vortexed for 15 seconds before and after heating in a water bath at 82.5°C for 5 minutes, and then centrifuged at 14,000 rpm for 2 minutes. The remaining steps followed the manufacturer’s directions. Extracted stool DNA was stored at -20°C prior to PCR testing.
Quantification of C. difficileshedding by qPCR
C. difficile DNA was analyzed from extracted stool DNAs with iQ SYBR Green Supermix in a 96-well plate performed at CFX96™ Real-Time PCR Detection System (Bio-Rad). Briefly, a PCR master mix was prepared with 23 μL aliquot containing 1 μL each of tcdB forward and reverse primers, 12.5 μL iQ SYBR Green Supermix, and 8.5 μL of H2O purified by Milli-Q Integral Water Purification System (Millipore Corporation, Billerica, MA). 2 μL of each sample was then added to each well filled with PCR master mix aliquot in 96-well. The PCR parameters were sequentially set for 3 stages: 1× cycle for 5 minutes at 94.0°C, 40 × cycle for 30 seconds each from 94.0°C and 55.0°C to72.0°C, 64 × cycle at 62.0°C for 15 seconds, and 1× cycle for hold at 25.0°C. Melt curve data collection and analysis were enabled. Copy numbers of unknown sample were extrapolated from the standard curve that was generated with the extracted DNA prepared from known C. difficile inocula. The sequence of tcdB forward primer was 5′-GGAGAGTCATCCAACTTATATG-3′; the sequence of tcdB reverse primer was 5′-CCACCAATTTCTTTTAATGCAG-3′.
The middle cross section of cecum and proximal cross section of colon were harvested from moribund mice or surviving mice at the end of the experiment. Tissues were fixed overnight with Bouin’s solution and stored in 70% ethanol until subsequently processed for Hematoxylin & Eosin (HE) staining at University of Virginia Research Histology Core. Slides were examined using a Leica DFC425 digital camera equipped microscope with Leica Application Suite Version 18.104.22.1688 imaging software (Leica Microsystems Inc., Buffalo Grove, IL 60089). Intestinal tissues were scored from 0 to 3 (with zero as normal and 3 as the worst pathologic score) under 5 categories: overall architecture, mucosal thickness, submucosal edema, inflammation, and exudates.
ELISA for IFNγ and TNFα
One set of infection experiments with A2AAR -/- (n=8) and their littermate A2AAR +/+ (n=8) mice were set aside for blood and tissue cytokine assays at the peak of infection (day 3) and also at recovery (day 7). Blood samples were collected in heparinized tube by cardiopuncture under sedation. Plasma samples were obtained by centrifuging blood at 10,000 rpm at room temperature for 20 minutes and stored at -80°C until further analysis. Upon euthanasia, cecal samples were harvested and stored at -80°C. ELISA was performed using Thermo Scientific Pierce Mouse IFNγ and TNFα kits (Rockford, IL) with slight modification of the manufacturer’s instruction. Briefly, cecal tissue was homogenized by grinding on dry ice and suspended in diluent reagent (0.2 mg/ml). Each homogenate or plasma sample was incubated with IFNγ antibody (for 1 hour) or TNFα antibody (for 2 hours) in 1:20 final dilution (200 μl) at room temperature. After washing, the sample was incubated with 100 μl of streptavidin-horseradish peroxidase (HRP) for 30 minutes. Subsequently, 100 μl of TMB substrate was added for another 30-minute incubation in the dark. The incubation was terminated with 100 μl of stop solution. Samples were immediately measured at 450 nm and 550 nm in Gen5 1.11.5 version in BioTek Spectrophotometer (Winooski, Vermont). Standard curves were established by plotting the average absorbance obtained for each standard known concentration (pg/ml). Cytokine amount in each sample was extrapolated from the standard curves.
Statistical analyses were conducted using GraphPad Prism Version 5.02 software. When mouse was either found dead or sacrificed due to severe distress, its last body weight recording was continuously plotted against the body weights of surviving mice. Differences between groups for the entire experimental period were analyzed by 2-Way ANOVA with Bonferroni post hoc testing. Survival curves were analyzed using Log-rank (Mantel-Cox) or Log-rank test for mortality trend.
A2AAR agonist reduced diarrhea and deaths in C. difficile- infected mice
Characterization of the A 2A AR agonist, ATL370, by radioligand binding (mean ± SD)
A2AAR - higha
Human (K i , nM)
61.1 ± 11.3
0.5 ± 0.2
3.8 ± 0.7
130.4 ± 44.4
Mouse (K i , nM)
65.3 ± 15.8
4.3 ± 2.8
84.2 ± 19.2
We next tested the effect of ATL370 at a higher dose but shorter duration of treatment, we used 10 ng/kg/min dosing given for 7 days (via Alzet pump). Similar to what was observed earlier, infected mice treated with vancomycin fared well for several days until discontinuation of vancomycin. Addition of ATL370 to vancomycin improved survival by 20%. Infected mice that received ATL370 alone had a 0% survival rate. Weight changes and clinical scores followed the same trend as the first study. Taken together, these data suggested that low dose ATL370 may be as beneficial as higher doses if given with vancomycin and confirmed that A2AAR agonist alone could exacerbate disease during acute infection.
Early or delayed administration of A2AAR agonist improved outcome of C. difficileinfection
Reduced vancomycin exposure further increased survival in the presence of A2AAR agonist in mice infected with C. difficile
The absence of A2AAR worsened C. difficileinfection in mice
The absence of A2AAR activation altered the inflammatory response during CDI in mice
Discussion and conclusions
Vancomycin is the drug of choice for severe CDI [27, 28]. However, its use has been associated with clinical recurrence of infection in up to 20% of cases, which has been attributed to antibiotic-induced changes of gut microbiota . In the mouse model of infection, recurrence of disease and late mortality has also been observed to occur in 40-60% of mice treated with vancomycin [24, 30]. Typically, mice treated with vancomycin remain well and only develop disease several days after cessation of the antibiotic. In our study, we demonstrated that the addition of A2AAR agonists during vancomycin treatment decreased recurrence and associated late mortality in mice infected with C. difficile. We then confirmed the role of A2AARs in CDI by showing that the complete absence of A2AAR activation exacerbated disease and alteration of inflammatory cytokine expression.
Intestinal epithelial cells have a very limited capacity for de novo adenosine synthesis . The villous cells can directly use absorbed dietary nucleosides but the cryptal cells depend on blood supply. Under normal adenosine homeostasis, extracellular adenosine concentrations can be lower than 1 μM [32, 33]. In response to cellular damage, adenosine concentrations are quickly elevated 100-fold higher in inflamed intestine due to ATP and adenosine secretion in inflammatory and other cell types. Control of inflammation and injury is thought to be secondary to A2AAR activation in intestinal tissue reperfusion injury  or experimental colitis . The A2AAR has been shown to inhibit neutrophil cytotoxic activities such as expression of β2-integrins , adhesion to endothelium , production of oxygen radicals [38, 39], degranulation , and production of TNF-α . Given intense intestinal inflammation, including neutrophilic tissue infiltration, noted in CDI in mouse , blockade or absence of A2AARs would, then, be expected to result in aggravation of disease. Indeed, in our study, A2AAR knockout mice had worse colitis (even in mice surviving infection) and more deaths from C. difficile infection compared to wild-type littermates suggesting that endogenous adenosine provides protection against infection through the A2AAR. A2AAR agonists added to 3 days of vancomycin treatment improved survival, clinical scores, and cecum histopathology compared to vancomycin alone when treatment was started at least one day after the animals were infected. This benefit was probably mediated by the anti-inflammatory effects of A2AAR activation counteracting the pro-inflammatory effects of C. difficile toxins.
Previous studies have shown that A2AAR activation with ATL 313 or inhibition of adenosine deaminase attenuated C. difficile toxin A-induced ileitis in mice [20, 21]. Myeloperoxidase activity, TNF-α production, cell death and histopathology were all noted to be reduced in ileal tissues treated with ATL 313. Recently, we showed that the A2AAR agonist, ATL370, decreased toxin A-induced secretion and epithelial injury in rabbit ileum and decreased KC (keratinocyte chemokine) and IL10 levels in mouse cecal tissues . In the current study, we demonstrate that deletion of A2AARs in mice, resulted in delayed but augmented expression of inflammatory cytokines, specifically IFNγ and TNFα during infection suggesting that initial inflammatory response is essential in controlling the disease. Indeed, the administration of ATL 370 alone resulted in greater and quicker mortality in most of the treated C. difficile-infected mice suggesting that A2AAR activation may inhibit the natural and beneficial early immune response initiated by infection. Furthermore, antibiotic treatment is essential to control clostridial burden and development of severe disease. This is consistent with what had been shown in a mouse model of sepsis where another A2AAR agonist, ATL 146e, was shown to reduce mortality in endotoxemia from LPS but not in E. coli septicemia unless antibiotic was also given . It is possible that adenosine is utilized by bacteria to enhance virulence as observed in other pathogens [43, 44]. Interestingly, although infected knockout mice had worse mortality than their infected wild-type littermates, 50% of these mice still survived in the absence of vancomycin treatment. Differences in intestinal flora (transgenic mice were bred in house) as well as other host factors may play a role in susceptibility to severe disease.
Our observation that A2AAR agonist alone (administered at the start of infection) worsened outcomes possibly due to inhibiting the natural immune response early on prompted us to investigate whether delaying A2AAR agonist treatment in addition to treating with vancomycin would improve outcomes. We showed that delaying the start of A2AAR agonist treatment post-infection was as good as starting A2AAR agonist at the same time as vancomycin administration suggesting benefit of A2AAR activation even at a later time during the course of infection and antibiotic treatment. While our study showed that antibiotic treatment is necessary to cure infection, we also demonstrated that overtreating with vancomycin may yield the worst outcomes. Reducing the antibiotic treatment to 2 days improved outcomes and adding A2AAR agonist to this regimen reduced the mortality associated with vancomycin treatment alone by 50%. These observations may have important ramifications if they translate to the clinical setting. If vancomycin (or antibiotic) treatment duration can be reduced by adding an A2AAR agonist, recovery of the gut microbiota may be facilitated and the recurrence of infection after antibiotic therapy may be improved considerably.
Although previous studies have shown that A2AAR activation confers significant protection against C. difficile toxin-induced ileitis and cecitis [20–22], protection against severe disease in the mouse model of infection seems limited. We recently reported that A2BAR inhibition or deletion, even in the absence of an anti-clostridial agent, improved outcome of CDI in mice . During infection, the clostridial bacteria are localized in the lumen and mucosal surface of the intestinal tract. The A2BAR is the predominant adenosine receptor in human intestinal epithelial cells  and thus, may have a greater role than A2AAR in mediating local tissue inflammation in response to C. difficile infection. However, A2AAR activity may be critical in controlling inflammatory response from immune cells recruited to the intestinal tissues and/or circulating immune cells during severe disease. More studies are needed to elucidate the interactions between different adenosine receptor subtypes during enteric infection.
In conclusion, in a murine model of CDI, vancomycin treatment resulted to reduced weight loss and diarrhea during acute infection, but was associated with high recurrence and late-onset death, with overall mortality being worse than untreated infected controls. Deletion of A2AARs in mice worsened disease from CDI. The administration of an A2AAR agonist reduced the late mortality associated with vancomycin use, suggesting a possible adjunctive benefit of A2AAR agonists in the management of CDI to prevent recurrent disease and improve survival.
We acknowledge Gina Calabrese, Edward van Opstal and Snjezana Zaja-Milatovic for their technical support and Robert Warren for reviewing the manuscript. The results of this work were partially presented at the 49th Annual Meeting of the Infectious Disease Society of America, Boston MA (Abstract #916). Oct. 19-23 2011. This study was supported by the National Institutes of Health/National Institute of Allergy and Infectious Diseases [U01 AI075526].
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