Ecto-5′-Nucleotidase (CD73) Regulates Host Inflammatory Responses and Exacerbates Murine Salmonellosis

Food-borne Salmonella spp., are a major cause of hospitalization and death. Adenosine, an important immune regulator of inflammation, limits tissue damage during infection. CD39 (nucleoside triphosphate dephosphorylase) combined with ecto-5′-nucleotidase (CD73) metabolizes ATP to adenosine. We studied the expressions of CD39 and CD73 in tissues, and T helper cells in mice after Salmonella infection and evaluated the role of CD73 in regulating immune responses and bacterial clearance in wild-type and CD73-deficient (CD73−/−) mice. Both CD39 and CD73 transcript levels declined in the infected wild-type mice. Compared to wild-type mice, tissues from infected CD73−/− mice had significantly higher expression of pro-inflammatory cytokines and reduced anti-inflammatory responses. CD73−/− mice were more resistant to infection and had a greater inflammatory responses and a significantly lower bacterial load in the liver compared to wild-type mice. Thus, CD73 expression attenuates inflammation during murine Salmonellosis and impairs immunity, leading to increased bacterial colonization and prolonged infection.

Treg cells regulate inflammation and contribute to the persistence of Helicobacter pylori infections 18,27 . Depletion of CD4 1 CD25 1 Treg cells enhances effector T cell activation and reduces pathogen burden during Leishmania major infection 28 . Similarly, other reports suggest a correlation between the increased persistence of Plasmodium falciparum with upregulation of TGF-b1, Foxp3, and CD4 1 CD25 1 Treg cells during human malaria infection 29 . We recently reported the important role of CD73 in modulating Th cell responses and H. pylori persistence 30 . Little is known, however, about the role of adenosine in controlling the host response to infection with Salmonella species. In the present study, we examined the expression of CD39 and CD73 in tissues and Th cells, including Treg cells, during murine Salmonella infection and evaluated the role of CD73 in regulating inflammation and bacterial burden using CD73-deficient mice.

Results
Regulation CD39 and CD73 expression following Salmonella infection. CD39 and CD73 transcript levels are upregulated during hypoxia and inflammation 31 , however, little is known about mRNA or protein expression in response to infection. Following oral Salmonella challenge in wild-type (C57BL/6) mice, the mRNA transcript levels of CD39 and CD73 sharply decreased in the liver and spleen (Fig. 1a, b). Similar decrease was also documented in the intestine (Fig. 1c). Recent evidence suggests the involvement of CD39 and CD73 in the control of CD4 1 Th function 24,32,33 . We therefore examined the surface expression of CD39 and CD73 in splenocytes after Salmonella whole cell lysate (WCL) treatment. Both CD39 and CD73 were abundantly expressed by uninfected or resting splenocytes and CD4 1 Th cells (Fig. 1d, e). Expression of both CD39 and CD73 was decreased in lymphocytes and CD4 1 Th cell populations when splenocytes were treated with Salmonella WCL (Fig. 1d, e, f). Taken together, these data suggest that murine tissues and Th cells express CD39 and CD73 to generate adenosine locally, and Salmonella infection leads to significant downregulation in both CD39 and CD73 expression.
Inhibition of CD73 enzymatic activity augments pro-inflammatory responses. To examine the contribution of CD73 to cytokine levels during Salmonella infection, murine splenocytes were treated with Salmonella-WCL in the absence or presence of APCP. Salmonella-WCL treatment increased the intracellular cytokines IL17A and IFN-c in splenocyte populations and CD41 cells and their expression was significantly augmented by CD73-inhibition (Fig. 2). Similarly, increased levels of IFN-c and IL17A were found in culture supernatants of Salmonella-WCL treated splenocytes, and after CD73 inhibition by APCP treatment these cells (Supplementary data: Fig. S1) produced significantly higher levels of the cytokines. Thus, in vitro CD73-inhibition increases Salmonella-WCL induced expression of pro-inflammatory cytokines. Since 59-AMP is cleaved by CD73 to generate extracellular adenosine; the specificity of CD73 with APCP was tested by measuring the pro-inflammatory cytokines in the supernatant from culture of Salmonella-WCL treated splenocytes in presence of 59-AMP with or without APCP (Fig.  S1c). Addition of 59-AMP in the culture suppressed the proinflammatory cytokines and, which was partially reversed by the addition of APCP. This, functional assay provided evidence that, splenocytes expressing CD73 generate adenosine that suppresses pro-inflammatory cytokines. It is known that adenosine acts via adenosine receptors expressed by immune and inflammatory cells that are upregulated upon activation 21,23 . Salmonella infected spleen tissues express more A 2a AR mRNA than A 2b AR (data not shown). To find if adenosine receptors were mediating the changes in cytokine responses, NECA, a non-selective adenosine agonist, was tested by measuring the pro-inflammatory cytokines in the supernatant from the culture of Salmonella-WCL-treated splenocytes in presence or absence of ZM241385, an A 2a AR antagonist (Fig. S1d).
NECA suppressed the pro-inflammatory cytokines induced by ST-WCL treatment and which was partially abrogated by the addition of ZM241385, confirming that the effect could be also mediated through adenosine receptors. These data confirm that extracellular adenosine produced by CD73 can act via adenosine receptors.
CD73-Deficient mice exhibited increased inflammatory responses following infection. To further investigate the role of CD73 in the control of inflammation in infected mice, we evaluated cytokine expression from splenocytes populations and CD4 1 Th cell populations of the infected wild-type and CD73 2/2 mice. Higher levels of intracellular pro-inflammatory cytokines, such as IFN-c, and IL17A, were observed in splenocytes isolated from the infected CD73 2/2 mice than in those isolated from wild-type infected mice (Fig. 3c, d). Similarly, responses at the transcript level for IFN-c and IL17A were significantly higher in spleens from infected CD73 2/2 mice (Fig. 3a, b).
Histologic examination of the liver 8 days post infection revealed mild to moderate hepatic damage in both strains. The liver from the CD73 2/2 mice exhibited moderately more inflammation, with aggregates of lymphocytes and macrophages extending to multiple foci throughout the parenchyma, and occasionally surrounded by small numbers of neutrophils (Fig. 4). Rarely within these areas, small foci of necrosis were observed. In both strains, there was evidence of hepatocellular injury accompanied by vesiculation, Kupffer cells swelling and edema, however the inflammatory infiltrate in the wild-type mice liver was less pronounced and centered around portal tracts. The liver tissues from CD73 2/2 mice also had significantly higher mRNA expression for the pro-inflammatory cytokines (IFNc, TNF-a, IL-1b) and inducible nitric oxide synthase (iNOS) (Fig. 4c). These findings indicate that the number of inflammatory-foci and pro-inflammatory responses in the liver were significantly increased in the absence of CD73 expression, consistent with the increased Th responses in the spleen (Fig. 3).
Anti-inflammatory responses following Salmonella infection. We next examined CD4 1 CD25 1 Treg cell subsets and their expression of CD39 and CD73 cells after infection (Supplementary data, Fig. S2). Salmonella infection slightly increased the percentage of Treg cells (CD4 1 CD25 1 ) in wild-type and CD73 2/2 mice while the percentage Treg expressing CD73 decreased significantly (Supplementary data, Fig. S 2c) after infection. Following infection of CD73 2/2 mice, in the absence of CD73 1 cells, Treg cells from these mice may not function properly. A previous report indicated that CD73 2/2 mouse-derived Treg cells are functionally deficient in regulating T effector cells 24 . Evaluation of splenic tissue and splenocytes for anti-inflammatory cytokine expression revealed no significant upregulation of IL-10 in infected CD73 2/2 mice compared with infected wild-type mice (Fig. 5). Moreover, IL-4, TGF-b1 and IL-13 mRNA response, which is also involved in regulating anti-inflammatory responses by the Treg cells, was significantly reduced in the infected CD73 2/2 mice.
CD73-deficient mice are more resistant to Salmonella infection than wild-type (CD73 1/1 ) mice. Since inhibition of CD73 by APCP enhanced cytokine responses following stimulation with Salmonella-WCL, the role of CD73 in the host response to infection was examined in CD73 2/2 mice. Both wild-type and CD73 2/2 mice were infected with variable doses of Salmonella, and body-weight was monitored. Wild-type mice lost significantly more bodyweight than CD73 2/2 mice following oral infection with 1 3 10 6 CFU/mice (Fig. 6). With an oral infective dose of 1 3 10 7 CFU, both groups of mice became sick, but CD73-deficent mice lost relatively less body weight than infected wild-type mice. In fact, throughout the Salmonella infection experiments, we observed that significantly more ST-infected wild-type mice displayed severe clinical signs than CD73-deficient mice (Supplementary data, S3, Table). CD73 gene deletion enhances clearance of Salmonella from infected tissues. Since CD73 gene deletion in mice confers resistance against ST infection and causes significantly enhanced pro-inflammatory responses in these tissues, we hypothesized that the adenosine-mediated regulation of Th cells would contribute to Salmonella persistence. To investigate this, an in vivo Salmonella infection study was conducted. Salmonella colonization in the liver was significantly lower in the CD73 2/2 mice than in the wild-type mice (Fig. 7). A separate infection experiment using a lower infection dose to evaluate long-term Salmonella colonization 20 days after infection revealed that CD73 2/2 mice effectively cleared most of the Salmonella, while wild-type mice had significantly higher   bacterial colonization in the liver and spleen on 13-dpi (Fig. 7b). Of note, infected wild-type mice became extremely sick by 13 days post infection and were euthanized to count bacterial load while CD73 2/2 mice recovered. We monitored infected CD73 2/2 mice until 20-dpi infection and bacterial count were performed.

Discussion
The control and clearance of intracellular infections require an appropriate host response by the coordinated action of innate and adaptive immunity. In the mouse model of typhoid, clearance of Salmonella requires production of IFN-c by CD4 1 T cells 10,11,13,34 and IL17 from Th17 cells 35 which in turn plays an important role in amplifying the inflammatory responses in the liver 36 and gut 14 . Again, the balance between CD41 Teff cell response and Treg cells can dictate the course of persistent infection 15 . Thus, the disruption of one of the mediators produced by Treg, adenosine, increased Th-1 and Th17 responses from infected CD73 2/2 mouse splenocytes. This notion was supported by the observation that splenocytes stimulated with Salmonella-whole cell lysate produced IFN-c and IL17A, and CD73-inhibition with APCP enhanced this response. The mRNA expression of IFN-c, TNF-a, and IL1-b, was significantly increased in the livers of CD73 2/2 mice in association with an increase in   inflammation. CD73-deficient mice also had better survival, lost significantly less body weight, and showed enhanced competence in eliminating Salmonella infection in the liver and spleen compared to wild-type mice.
In addition to the generation of effective anti-bacterial pro-inflammatory responses, infected CD73 2/2 mice had increased liver iNOS mRNA expression. The increase in iNOS is consistent with a report suggesting that the engagement of A 2B AR agonists suppresses NO production in LPS-treated RAW 264.7 macrophages 37 . Therefore, adenosine may regulate NO production to influence innate host responses. Further studies examining the role of CD73/adenosine pathways on NO production by macrophages is needed to determine whether these regulatory effects altered Salmonella killing by these cells.  IFN-γ * *  Because CD39 and CD73 are rate-limiting factors for extracellular adenosine generation, the ability of Treg cells to synthesize adenosine from ATP and ADP can contribute significantly to the suppression of effector cells during infection-induced inflammation. In Th cells, the expression of CD39 and CD73 by CD4 1 CD25 1high Treg cells and the presence of A 2A AR on activated effector Th cells provide an immunosuppressive loop, whereby Treg generate adenosine that inhibits the function of effector Th cells 24,30,32,38 . Previous findings that Treg cells prepared from mice lacking CD73 fail to regulate inflammation compared to wild-type Treg cells further support the notion that  adenosine contributes to the pool of mediators required for optimal Treg cell function 26,30 . In the present study, we observed reduced production of anti-inflammatory cytokines (IL-10/IL-4/TGF-b1) in infected CD73 2/2 mouse tissues, which further suggests that Treg cells from these mice do not produce sufficient levels of IL-10 and TGF-b1 to attenuate the pro-inflammatory response.

IFN-γ IL17A
In addition to our findings that CD73 regulates immunity and exacerbates Salmonellosis, both adenosine A 2A AR and A 2B AR are important for governing the morbidity and mortality during poly-microbial sepsis and infection 20,[39][40][41] . Together with our observations, these reports support the notion that adenosine is a significant regulator of disease in mice during infection.
The complexity of adenosine function is illustrated by Hasko et al 42 who reported an opposite effect of CD73 during sepsis. In addition, Kolachala el al 43 showed that Salmonella infection of A 2B AR 2/2 mice led to increased mortality compared to the wild-type counterpart. It is important to note that the pathogenesis of polymicrobial sepsis and different Salmonella models can vary in many ways due to the microbiota in our respective housing facilities. Further, some bacteria adapt to exploit immunosuppressive pathways to increase their own survival 44 , leading to the possibility that adenosine favors the survival of a pathogen if it is present at the correct time or context. In fact, Mohamed et al recently showed that CD73 2/2 mice are protected from Toxoplasma gondii-induced mortality due to impaired parasite differentiation in the host 45 . The present finding that CD73 suppresses inflammation and impairs bacterial clearance is consistent with a previous report 30 showing that CD73 inhibition or A 2A ARdeletion 18 augments pro-inflammatory responses in human Th cells as well as increases gastritis in an animal model of H. pylori infection. Moreover, CD73 may mediate noncatalytic functions related to costimulation and/or adhesion 46 . However, the increase in inflammation in infected CD73 2/2 mice and the effects of the enzymatic inhibitor APCP observed in the current study favors the hypothesis that the absence of CD73 enhances T cell activation by compromising the synthesis of adenosine rather than disruption of activation signals.
In our study we observed Salmonella infection reduced the expression of both CD39 and CD73 in a tissue, suggesting the host attempts to favor adaptive responses to clear infection. This response might be essential to avoid the suppressive effects due to adenosine generation during T cell activation 38 . While CD39 and CD73 expression on Treg may decrease in response to infection, the mechanism accounting for this remains unclear. One possibility is that CD73 expression is modulated by the cytokine milieu present during activation by Salmonella infection, for example, the decrease in TGF-b1 would remove a potent stimulus for CD73 expression 38,47 . However, if this effect is designed to control bacterial burden, it is not as dramatic as the CD73-deficient mice in which the absolute  disruption of this pathway decreases bacterial burden and increases survival. This is due to the fact that CD73 expression by effector Th cells and other cells might also contribute to the adenosine pool, which serves to limit host responsiveness. In turn, this pool would inhibit the anti-microbial responses and favor infection. In summary, expression of CD39 and CD73 by Th cells and the enrichment of both enzymes in murine Treg cells suggest that Th cells contribute to local adenosine accumulation and the control of inflammation. Diminished adenosine generation in APCP-treated cells or CD73-deficient mice was associated with an enhanced pro-inflammatory response that reduced the levels of Salmonella colonization upon challenge. These observations support the notion that adenosine production and the ability of adenosine to limit inflammation favors bacterial survival, thereby broadening the impact of infection.
Mice. Wild-type, C57BL/6 (CD73 1/1 ) black mice were purchased from The Jackson Laboratory (Bar Harbor, ME). CD73-deficient (CD73 2/2 ) mice inbred onto the C57BL/6 background were kindly provided by Dr. Linda Thompson 48 . Salmonella challenge of mice. To establish a primary Salmonella infection, mice were inoculated by gavage with Salmonella LT-2 strain containing 1 3 10 6 to 1 3 10 7 CFU of the bacteria. Prior to infection, cage-bedding was exchanged for at least 2 weeks between wild-type (C57BL/6) and CD73 2/2 mice by mixing soiled beddings (containing fecal materials) from two sources and then redistributing it with the fresh bedding materials. Bedding exchange was performed to equilibrate the colonization of the commensal gut flora between both wild-type and CD73 2/2 mice, which was believed to generate specific gut immune response 49 . In addition, to ensure equal infection (dose), prior to infection, all mice were fasted for approximately 6 hours. Metal wire-flooring was used in the cage for a maximum of 3 days to prevent coprophagia. Mice were monitored daily for clinical signs of disease and body weight was measured. Infected mice that became severely sick were euthanized as par FDA-CFSAN-IACUC guidelines. Mice that became suddenly sick or died within 3 days of infection without any significant changes in the body weight were not included in the study.  Histopathology of liver samples. Portions of liver were fixed in Bouin's solution (Ricca Chemical, Arlington, TX), washed with ethanol, embedded in paraffin, cut into 3-to 5-mm sections, and stained with hematoxylin and eosin (H&E). All H&E stained slides were scanned and digitally stored using the Nanozoomer (Hamamatsu, Japan) with their NDP-view software (Hamamatsu). Liver inflammation was quantified by counting the number of inflammatory foci defined as a region with mononuclear immune cell infiltrate at 103 magnification.
Assessment of Salmonella colonization in liver. For quantitative measurement of Salmonella colonization, a portion of liver tissue was homogenized in PBS, and replicate serial 10-fold dilutions were plated in LB agar plates to determine bacterial numbers.
In some experiments 59-AMP (0.5-50 mM) or NECA (500 nM) or ZM241385 (500 nM) was also used in a similar splenocytes stimulation assay as described earlier 18,25 . Supernatant and cells were collected after 24 h of incubation and used for ELISAs or multiplex bead array.
Surface and intracellular cell staining and flow cytometry. Isolated splenocytes were washed and resuspended in PBS. Cells were placed on ice and labeled with fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD4 1 (BD Pharmingen, CA), allophycocyanin (APC)-conjugated anti-mouse CD25 (BD Pharmingen), phycoerythrin (PE) conjugated anti-mouse CD73 (BD Pharmingen) and PE-Cy7conjugated anti-mouse CD39 (eBiosciences, CA). Intracellular staining of splenocytes was also performed after surface staining in some experiments. For intracellular staining, cultured cells were collected and stimulated with leukocyte activation cocktail (PMA/ionomycin), with BD GolgiPlug (BD Biosciences) according to the manufacturer's protocol, for 4 h. Cells were incubated with FcBlock, stained with anti-CD4 (BD Biosciences), permeabilized, and stained with anti-IL-17A (eBiosciences), or anti-IFN-c (eBiosciences), using a Cytofix/Cytoperm kit (BD Biosciences). Cells were also stained with their respective isotype controls as recommended in the manufacturer's protocols for adjusting gates. Samples were assayed with a BD FACSCalibur flow cytometer, and data were analyzed with FlowJo (TreeStar) software.
Cytokine assays. IFN-c and IL-10 concentrations in the supernatants of Salmonella WCL-stimulated cultures were measured by ELISA according to the manufacturer's protocol (BD Biosciences, CA). Similarly IL17A concentration was measured by ELISA according to the manufacturer's protocol (eBiosciences). For some experiments supernatants collected from the culture assays were assayed for Th1 cytokines by use of a multiplex bead array (Bio-Rad, CA) and were analyzed using the Bioplex workstation and associated software (Bio-Plex Manager, version 6.1; Bio-Rad).
RNA extraction and Real-time RT-PCR. Total RNA was extracted from liver, spleen, and splenocytes using Qiagen kits (Qiagen, Valencia, CA). In each case RNA was reverse-transcribed to yield cDNA using the RT 2 First Strand kit (Qiagen). Transcripts were measured by Real-time RT-PCR and performed in CFX96 Real-Time System BioRad (Irvine, CA) using RT 2 SYBR Green qPCR and RT 2 qPCR primers (IFN-c: PPM03121A, TNF-a: PPM03113F, IL-1b: PPM03109E, IL-17A: PPM03023A, IL-13: PPM03021A, IL-10: PPM03017B, IL-4: PPM03013E, TGF-b1: PPM02991A and iNOS: PPM02928B) from SABiosciences Corporation-Qiagen Company (Frederick, MD). The levels of RNA for the target sequences were determined by melting curve analysis using the Bio-Rad CFX manager software (Irvine, CA). Normalized levels of each mRNA were determined using the formula 2 (Rt -Et) , where Rt is the threshold cycle for the reference gene (GAPDH: PPM02946E, Qiagen) and Et is the threshold cycle for the experimental gene (DDC T method) 50 . Data are expressed as arbitrary units.
Statistical analysis. Results are expressed as mean 6 SEM. Data were compared by Student's t test (unpaired) or AVOVA, and results were considered significant if p values were less than 0.05.