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Dysregulation of the inflammatory response to the parasite, Toxoplasma gondii, in P2X7 receptor-deficient mice
International Journal for Parasitology 41 (2011) 301–308
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Dysregulation of the inflammatory response to the parasite, Toxoplasma gondii, in P2X7 receptor-deficient mice Catherine M. Miller a, Alana M. Zakrzewski a, Rowan J. Ikin a, Nicola R. Boulter a, Marilyn Katrib a, Michael P. Lees a, Stephen J. Fuller b, James S. Wiley b, Nicholas C. Smith a,⇑ a b
Institute for the Biotechnology of Infectious Diseases, University of Technology, Sydney, Broadway, NSW 2007, Australia Sydney Medical School, Nepean Campus, Nepean Hospital, The University of Sydney, Penrith, NSW 2751, Australia
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Article history: Received 26 September 2010 Accepted 4 October 2010 Available online 31 October 2010 Keywords: Toxoplasma gondii Apicomplexa P2X7 receptor Nitric oxide IL-10 Immunopathology
a b s t r a c t The P2X7 receptor (P2X7R) is a two transmembrane receptor that is highly expressed on the surface of immune cells. Loss of function polymorphisms in this receptor have been linked to increased susceptibility to intracellular pathogens. P2X7R gene knockout (P2X7R/; on a C57Bl/6J background), C57Bl/6J and BALB/c mice were infected with the avirulent ME49 strain of the intracellular parasite, Toxoplasma gondii, and susceptibility determined by monitoring weight loss. P2X7R/ mice lost significantly more weight than C57Bl/6J mice from day 8 p.i. C57Bl/6J, in turn, lost significantly more weight than BALB/c mice. Thus, by day 10 p.i., P2X7R/ mice had lost 5.7 ± 0.7% of their weight versus 2.4 ± 0.6% for C57Bl/6J mice, whereas BALB/c mice had gained 1.9 ± 0.5%; by day 12 p.i., P2X7R/ mice had lost 15.1 ± 0.6%, C57Bl/6J had lost 10.1 ± 0.8% and BALB/c had lost 4.8 ± 0.8% of their weight. Neither parasite burden nor liver pathology was greater in the P2X7R/ mice than in C57Bl/6J mice but BALB/c mice had significantly smaller numbers of parasites and less pathology in their livers than these strains. Absence of the P2X7 receptor did not affect IFN-c, IL-12, IL-1b, monocyte chemoattractant protein-1 (MCP-1) or TNF production. However, both P2X7R/ and C57Bl/6J mice produced more IL-1b and TNF than BALB/c mice. There was one important point of differentiation between the P2X7R/ and C57Bl/6J mice, namely the significantly enhanced and prolonged production of nitric oxide, accompanied by delayed production of IL-10 in the P2X7R-deficient mice. Ó 2010 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction Toxoplasma gondii is a protozoan parasite that is able to infect and survive in cells of the monocyte/macrophage lineage; it possesses a range of sophisticated immune evasion strategies (Montoya and Liesenfeld, 2004; Aliberti, 2005; Miller et al., 2009a). It is prevalent in humans and animals worldwide with human infection rates varying from 10% to 90% depending on factors such as animal exposure and dietary habits (Montoya and Liesenfeld, 2004). Overall, it infects approximately one-third of the world’s population and can cause serious disease in pregnant women and immuno-compromised hosts. Pathology is very rare in healthy immuno-competent people, although there are significant numbers of cases of lymphadenopathy or ocular problems around the world every year (Montoya and Liesenfeld, 2004). Acute infection is characterised by the proliferation of rapidly dividing tachyzoites ⇑ Corresponding author. Address: Institute for the Biotechnology of Infectious Diseases, University of Technology, Sydney, P.O. Box 123, Broadway, NSW 2007, Australia. Tel.: +61 2 9514 4013; fax: +61 2 9514 4201. E-mail address: [email protected] (N.C. Smith).
that stimulate production of high levels of IL-12 and IFN-c by the cells of the innate immune system. Both cytokines play a central role in the development of resistance to T. gondii. Macrophages, together with dendritic cells and neutrophils, are an important source of IL-12 early in infection (Aliberti, 2005; Miller et al., 2009a). IL-12 activates natural killer (NK) cells and, later, T cells to produce IFN-c, which in turn promotes classical activation of macrophages via a positive feedback loop (Miller et al., 2009a). Classically activated macrophages play a key role in the control of intracellular parasite growth through the production of inflammatory cytokines and nitric oxide, and trigger apoptosis of the infected cell and stage conversion of the parasite from tachyzoite to bradyzoite (Robben et al., 2004; Aliberti, 2005). However, an uncontrolled response leads to severe immunopathology (Miller et al., 2009a). Signals from surrounding cells or the macrophages themselves down-regulate macrophage activation and lead to the production of anti-inflammatory cytokines such as IL-10 and transforming growth factor-b (TGF-b). This regulation is central to controlling infection, as knockout mice lacking either of these two cytokines are more susceptible to inflammatory pathologies than wild type mice (Mosser, 2003); IL-10 plays a particularly
0020-7519/$36.00 Ó 2010 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijpara.2010.10.001
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significant role in preventing immunopathology in T. gondii infection (Gazzinelli et al., 1996; Suzuki et al., 2000). Recently, another factor has emerged as an important component of the inflammatory response to intracellular pathogens, namely the P2X7 receptor (P2X7R). Activation of monocyte and macrophage P2X7R has been shown to kill intracellular Mycobacterium, Chlamydia and Leishmania spp. (Molloy et al., 1994; Lammas et al., 1997; Kusner and Adams, 2000; Coutinho-Silva et al., 2001, 2003; Fairbairn et al., 2001; Stober et al., 2001; Placido et al., 2006; Darville et al., 2007; Chavez et al., 2009). The P2X7R gene is highly polymorphic and loss-of-function polymorphisms reduce control of Mycobacterium tuberculosis. Furthermore, inheritance of the 1513A>C single nucleotide polymorphism has been associated with susceptibility to extrapulmonary tuberculosis in humans (Saunders et al., 2003; Fernando et al., 2005, 2007; Shemon et al., 2006; Nino-Moreno et al., 2007). The P2X7R is an ATP-gated cation channel belonging to the P2X family of receptors. It is widely expressed by cells of the immune system (Chen and Brosnan, 2006). Brief exposure to ATP leads to an influx of Ca2+ and Na+ and an efflux of K+, while prolonged or repeated exposure causes the formation of large pores in the membrane of the cell that allows the passage of molecules up to 314 Da in size (Wiley et al., 2001). The main source of the ATP is extracellular ATP (eATP) released from activated platelets, endothelial cells, nerve cells and antigen-stimulated T cells, and from stressed or dying cells during an inflammatory response (Aga et al., 2004; Tsukimoto et al., 2006). Stimulation of the P2X7R is regarded as a key process in the inflammatory response to infection as it potentiates the release of various pro-inflammatory mediators such as IL-1b, TNF and nitric oxide, activates phospholipase D leading to phagosome/lysosome fusion, and induces cell death through apoptosis and/or necrosis (Coutinho-Silva et al., 2007; Lenertz et al., 2009). It has been reported recently that activation of the P2X7R by ATP can mediate killing of T. gondii (Correa et al., 2010; Lees et al., 2010) via, potentially, either apoptosis (Lees et al., 2010) or formation of phagolysosomes accompanied by generation of reactive oxygen species (Correa et al., 2010). These observations, coupled with the fact that many pathways involved with P2X7R activation are also typical of innate Th1 responses, makes it conceivable that the P2X7R has an important role in the induction of the immune response to T. gondii. In this study we demonstrate that mice lacking a P2X7 receptor (P2X7R/) are more susceptible to T. gondii infection than mice with half-functional and fully functional P2X7 receptors and this is associated with an apparent lack of regulation of the inflammatory response.
2. Materials and methods 2.1. Mice All animal research was performed with the approval of the University of Technology Sydney (UTS; Australia)/Royal North Shore Hospital Animal Care & Ethics Committee Protocols UTS/ RNSH 0611-042A and UTS ACEC 2008-03. Pathogen-free, 6–8 week old C57Bl/6J and BALB/c mice were obtained from the Gore Hill Research Laboratories (Sydney, Australia) or Animal Resource Centre (Perth, Western Australia). P2RX7 gene-deleted mice (P2X7R/) on a C57Bl/6J background (back-crossed for at least seven generations) were originally provided by Pfizer, Inc. (Ann Arbor, MI, USA) and were subsequently bred at the Gore Hill Research Laboratories and the Ernst Facility (UTS, Australia). C57BL/6 mice have a proline to leucine polymorphism at amino acid 451 in the C-terminal tail of the P2X7R that has various effects on P2X7R function (Adriouch et al., 2002; Le Stunff et al., 2004; Asward and Dennert, 2006; Hong et al., 2009; Suadicani et al., 2009; Yip et al., 2009).
Thus, we also included comparative analyses of BALB/c cells in our investigations, as this strain has fully functional P2X7R (Adriouch et al., 2002; Le Stunff et al., 2004; Asward and Dennert, 2006; Hong et al., 2009; Suadicani et al., 2009; Yip et al., 2009) and is also known to be more resistant to T. gondii than C57BL/6J mice (Araujo et al., 1976; Johnson, 1984; McLeod et al., 1989; Suzuki et al., 2000). Inclusion of the BALB/c infections in our analyses was important to internally define a resistant phenotype; thus, we were looking for differences between P2X7R/ mice and C57Bl/6J mice that were comparable to differences observed between C57Bl/6J mice and BALB/c mice, a recognised susceptible versus resistant pairing (Araujo et al., 1976; Johnson, 1984; McLeod et al., 1989; Suzuki et al., 2000). Lack of the P2RX7 gene in the knockout mice was confirmed by PCR using the following primers: For 50 -CTATCTCTCCACGACT CACCCCC-30 and Rev 50 -TATAATCCCGGGAGGGA TACTTGAAGCCAC TGTAC-30 (Adriouch et al., 2002). Lack of P2X7R function in splenocytes from uninfected P2X7R/ mice was also confirmed by measuring ATP-induced ethidium uptake as described previously (Lees et al., 2010). 2.2. Parasites and infections Tachyzoites of the T. gondii ME49 strain were obtained from the American Type Culture Collection (Manassas, VA, USA) and subsequently maintained by continuous passage in Vero cells (using RPMI from Invitrogen and 2% heat inactivated newborn calf serum) at 37 °C in a 5% CO2 incubator. Parasites were harvested from freshly lysed cultures, passed through a 26 G needle and concentrated by spinning at 500g. The pellet of tachyzoites was resuspended in sterile 0.9% saline. Parasites were counted using a Neubauer haemocytometer and diluted in sterile 0.9% saline to the required dose for injection. i.p. injection of tachyzoites was favoured for our model rather than oral infection with brain cysts because estimation of the infective dose of tachyzoites is more reliable than that of bradyzoites since each tissue cyst contains varying numbers of bradyzoites. Furthermore, the dissemination patterns following oral infection of mice with tissue cysts can be inconsistent (Boyle et al., 2007) and, ultimately, both infection routes result in similar immune responses, with i.p. infection leading to faster, more reproducible effects (Fouts and Boothroyd, 2007). To assess relative susceptibility to T. gondii infection, male mice of each strain were infected i.p. with 500 tachyzoites of T. gondii ME49 and monitored daily for clinical signs of T. gondii infection – weight loss, ruffled fur, hunched posture, lethargy and morbidity – for up to 3 weeks. Mice that showed signs of morbidity or had lost >20% of starting weight were euthanased by terminal anaesthesia as per Animal Care & Ethics Protocol stipulations. In total, 27 P2X7R/ mice, 27 C57Bl/6J mice and 28 BALB/c mice were infected over the course of three separate experiments. A total of 14, 13 and 12 uninfected P2X7R/, C57Bl/6J and BALB/c mice, respectively, served as uninfected controls. To identify aspects of the innate immune response that may be implicated in the increased susceptibility of P2X7R/ mice to T. gondii infection, mice were also euthanased at set time points. Two experiments were conducted where six mice of each strain were infected i.p. with 500 T. gondii ME49 tachyzoites and three from each strain were euthanased on days 8 and 12 p.i. In a third experiment, 12 mice from each strain were infected i.p. with 500 T. gondii ME49 tachyzoites and six from each strain were euthanased on days 8 and 12 p.i. 2.3. Assessment of parasite burden and liver pathology Relative parasite burdens were assessed in liver sections using immunohistology with a rabbit anti-T. gondii polyclonal antibody
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(RayBiotech, Inc., Norcross, GA, USA). Unstained formalin-fixed, paraffin-embedded sections of liver (4 lm thick) were de-paraffinised and antigen retrieval performed by heating in 10 mM citrate buffer pH 6.5. Immunoperoxidase staining was then performed using anti-rabbit secondary antibodies in an ABC staining system (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) according to the manufacturer’s instructions. Controls included incubating with no primary antibody or an unrelated rabbit IgG. Slides were dehydrated, mounted and examined using a light microscope and the number of parasitophorous vacuoles containing tachyzoites or tachyzoite antigen counted in five fields of view under 10 objective by two independent investigators. The livers of all animals were removed and assessed visually for signs of necrosis. Livers were scored according to the following criteria: (0) normal colour, no visible necrosis; (1) slight change in colour, no visible necrosis; (2) change in colour with moderate lesions; (3) change in colour with severe necrosis. Sections of formalin-fixed, paraffin-embedded livers (4 lm thick) from two infected and one uninfected mouse per strain on each day of infection were also examined for areas of necrosis and inflammation following staining with H & E. Sections were scored according to the following criteria. Cellular infiltrate: (0) absent; (1) one focal infiltrate; (2) multifocal infiltrates (<10 per field); 3-diffuse infiltrate (>10 per field). Necrosis: (0) no visible necrosis; (1) small areas of necrosis; (2) moderate levels of necrosis (<50%); (3) marked necrosis (>50%). Five fields of view using the 10 objective were scored per section by two independent investigators blinded to the strain of mouse and infection status. 2.4. Isolation and ex vivo culture of peritoneal exudate cells Peritoneal exudate cells were recovered by rinsing the peritoneal cavity with 6 ml of sterile PBS, pelleted at 500g, then resuspended in RPMI 1640 containing 10% newborn calf serum and cultured in six well plates at 37 °C/5% CO2. After 2–3 h incubation, non-adhered cells were removed by washing with RPMI and adhered cells (previously determined to be 89–91% macrophages; Miller et al., 2009b) were removed by scraping. Cells were counted, adjusted to a concentration of 1 106/ml and replated into 48 well plates. Cells were incubated at 37 °C/5% CO2 for 48 h before supernatants were removed and stored at 20 °C until analysis. Supernatants were assayed for nitric oxide as described below.
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Nitric oxide concentrations were determined using the Griess assay as described previously (Miller et al., 2000). Briefly, nitric oxide produced by adhered peritoneal exudate cells was measured by mixing equal amounts of supernatant with Griess reagent (1% sulfanilimide in 2.5% H3PO4 and 0.1% napthylethylenediamine dihydrochloride in 2.5% H3PO4 mixed in a 1:1 ratio just prior to assay). Absorbance was measured at 540 nm and the amount of nitrite present was determined by comparing against a standard curve generated with known concentrations of sodium nitrite. 2.6. Statistical analyses The statistical significance of differences between groups was determined using a protected Students t-test coupled to a oneway ANOVA. A P value of <0.05 was considered significant. 3. Results 3.1. P2X7R/ mice are more susceptible to acute T. gondii infection than C57Bl/6J or BALB/c mice Susceptibility of P2X7R/ mice to acute T. gondii infection was assessed relative to susceptibility of C57Bl/6J mice and BALB/c mice by infecting mice with T. gondii ME49 strain and monitoring the course of infection. Mice were euthanased when they showed signs of morbidity or had lost >20% of starting weight. All infected mice exhibited clinical signs of infection and lost weight relative to uninfected mice of the same strain (Fig. 1). P2X7R/ mice lost significantly more weight than C57Bl/6J, which lost significantly more weight than BALB/c mice, beginning on day 8 p.i. Thus, by day 10 p.i., P2X7R/ mice had lost 5.7 ± 0.7%of their body weight versus 2.4 ± 0.6% for C57Bl/6J mice, whereas BALB/c mice had gained 1.9 ± 0.5%; by day 12 p.i., P2X7R/ mice had lost 15.1 ± 0.6%, C57Bl/6J had lost 10.1 ± 0.8% and BALB/c had lost 4.8 ± 0.8% of their body weight. Moreover, P2X7R/ mice succumbed to infection earlier so that, by day 18 p.i., 52% of the knockout mice had to be euthanased, whereas only 26% of the C57BL/6J and none of the BALB/c mice had to be euthanased. By day 21 p.i., 74% of the P2X7R/ mice had to be euthanased compared with 37% of C57Bl/6J and 36% of BALB/c mice.
2.5. Immunological assays Splenocytes were cultured at 5 106 cells/ml for 72 h without stimulation or with 50 lg/ml of T. gondii soluble extract or anticlusters of differentiation 3 (CD3; 2 lg/ml) plus phorbol 12-myristate 13 acetate (PMA) (25 ng/ml). Concentrations of the cytokines IL-12, IFN-c, IL-1b and IL-10 in serum and splenocyte medium were measured by immunoassay using matched pairs of anti-cytokine antibodies purchased from BD Pharmingen (San Diego, CA, USA) according to the manufacturer’s instructions. Recombinant cytokines of known concentration were used to generate standard curves. Levels of the pro-inflammatory cytokines, monocyte chemoattractant protein-1 (MCP-1) and TNF were measured using a BD Cytometric Bead Array kit (San Diego, CA, USA) according to the manufacturer’s instructions. Briefly, the serum samples and test standards supplied were mixed with cytokine capture beads and phycoerythrin (PE)-conjugated antibodies, and incubated for 2 h in the dark. After washing away unbound sample and reagent, fluorescence was measured in individual standards and samples on a BD LSR-II flow cytometer. Following data acquisition, standard curves were generated and cytokine amounts quantified using FCAP Array, version 1.0.1 (BD, San Diego, CA, USA).
Fig. 1. Weight loss caused by infection with Toxoplasma gondii is exacerbated in mice lacking P2X7 receptor (P2X7R) function. BALB/c, C57Bl/6J and P2X7R/ mice were infected i.p. with 500 tachyzoites of T. gondii ME49 strain and weighed daily. Cumulative weight loss of P2X7R/ mice was significantly greater than that of C57Bl/6J mice which, in turn, was significantly greater than that of BALB/c mice on each of days 8–14 p.i. (P < 0.05, protected Students t-test coupled to a one-way ANOVA; n = 27 for infected P2X7R/ and C57Bl/6J mice and n = 28 for BALB/c mice; n = 14, 13 and 12 for uninfected P2X7R/, C57Bl/6J and BALB/c mice, respectively). B-U = uninfected BALB/c, B-I = infected BALB/c, C-U = uninfected C57Bl/6J, CI = infected C57Bl/6J, P-U = uninfected P2X7R/ and P-I = infected P2X7R/ mice.
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Fig. 2. Parasite burden, gross liver pathology and histopathology in Toxoplasma gondii ME49-infected mice are not affected by the absence of the P2X7 receptor (P2X7R). Parasite burden in livers of BALB/c, C57Bl/6J and P2X7R/ mice (A) was determined by immunohistochemistry as detailed in the Materials and methods. Gross liver pathology (B) was assessed visually by scoring between 0 and 3 with a score of 0 indicating no visible necrosis and 3 indicating severe necrosis. Histopathology was assessed by examining H & E stained slides for areas of inflammation and necrosis ((C–E) pictures shown are from single mice and are representative of all mice examined in the groupfilled arrows indicate areas of cellular infiltrate and unfilled arrows indicate areas of necrosis). Levels of cellular infiltrate (G) and necrosis (H) were scored; a score of 0 indicating no inflammation or necrosis was evident while a score of 3 indicates high levels of infiltrate or necrosis. Scores are presented as mean ± standard error, n = 6 for gross liver pathology, n = 2 for histopathology and parasite burden. aIndicates where the score is significantly different from the score for BALB/c mice (P < 0.05, protected Students t-test coupled to a one-way ANOVA). KO, knockout.
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3.2. P2X7R/ and C57Bl/6J mice exhibit higher parasite burdens and extended liver pathology compared with BALB/c mice On days 8 and 12 p.i., parasite burden and liver pathology were assessed (Fig. 2). Cells containing T. gondii tachyzoites or antigen in liver sections were identified with an anti-T. gondii antibody and immunoperoxidase staining. Parasite burden and liver pathology were essentially identical in P2X7R/ and C57Bl/6J mice (Fig. 2A-H). However, significantly fewer areas containing parasites were identified in sections from BALB/c mice than in either C57Bl/ 6J mice or P2X7R/ mice on day 12 p.i. (Fig. 2A). Likewise, less gross pathology was evident in livers from BALB/c mice on both days 8 and 12 p.i., although the differences were more pronounced on day 12 p.i. There was little change in the scores for BALB/c mice on day 12 p.i. compared with day 8 p.i. but both C57Bl/6J and P2X7R/ mice showed a marked increase in pathology from day 8 p.i. to day 12 p.i. (Fig. 2B). Microscopic analysis of H & E stained sections from all three strains of mice confirmed these observations. Levels of cellular infiltrate and necrosis were similar in all three strains of mice on day 8 p.i. but, again, differences were quite pronounced on day 12 p.i. (Fig. 2C–G). Sections from infected P2X7R/ and C57Bl/6J mice (Fig. 2E–G) showed higher levels of infiltrate and larger areas of necrosis (Fig. 2E, F, and H) compared with sections from BALB/c mice (Fig. 2D, G, and H). No areas of cellular infiltrate or necrosis were seen in sections from uninfected mice (Fig. 2C, G, and H). 3.3. The IFN-c response to T. gondii infection is normal in mice with reduced or no P2X7R function IFN-c is crucial for resistance to T. gondii infection. Therefore, levels of this cytokine were assessed in serum from all three strains of mice on days 8 and 12 p.i. Levels of IFN-c were significantly elevated (P < 0.05) in infected mice of all three strains compared with uninfected mice of the same strain on both experimental days (Fig. 3A). All three strains of mice also showed a similar pattern of IFN-c production with a peak on day 8 p.i. followed by a decline in levels on day 12 p.i., although there were slight differences in amounts produced between the three strains (Fig. 3A). Levels of IFN-c were also assessed in supernatants from splenocytes cultured without antigenic stimulation (Fig. 3B). Again, a similar pattern of IFN-c production was seen amongst the three strains of mice with a peak on day 8 p.i. followed by a decline on day 12 p.i. There was no significant difference in IFN-c levels amongst the three strains of mice, either in serum (Fig. 3A) or splenocytes, whether stimulated with T. gondii antigen or PMA/anti-CD3 (data not shown) or not stimulated (Fig. 3B). 3.4. Production of pro-inflammatory cytokines is unaltered in mice lacking P2X7 receptor function compared with wild type mice Classical activation of macrophages results in the production of pro-inflammatory cytokines such as IL-12, IL-1b, MCP-1 and TNF. Levels of these cytokines were measured in the serum of infected mice from all three strains on days 8 and 12 p.i. (Fig. 4). Levels of IL-12 (Fig. 4A) were significantly elevated (P < 0.05) in infected mice of all three strains compared with uninfected mice of the same strain on both experimental days (Fig. 4A). All three strains of mice also showed a similar pattern of IL-12 production, with a peak on day 8 p.i. followed by a decline in levels on day 12 p.i., although there were slight differences in amounts produced between the three strains (Fig. 4A). There were significant increases (P < 0.05) in the levels of IL-1b (Fig. 4B), MCP-1 (Fig. 4C) and TNF (Fig. 4D) compared with uninfected mice on day 8 p.i. in C57Bl/ 6J and P2X7R/ mice and, subsequently, levels of IL-1b (Fig. 4B) and MCP-1 (Fig. 4C) declined significantly by day 12 p.i. whereas
Fig. 3. The IFN-c response to Toxoplasma gondii ME49 strain is normal in mice lacking P2X7 receptor (P2X7R) function. BALB/c, C57Bl/6J and P2X7R/ mice were infected i.p. with 500 tachyzoites of T. gondii ME49, euthanased on days 8 and 12 p.i. and serum collected via cardiac puncture. Splenocytes were cultured in vitro for 3 days before the supernatant was collected. Levels of IFN-c in serum (A) and spleen cell supernatant (B) were assayed by ELISA. Results are presented as mean ± standard error of one experiment where n = 6 infected mice per strain. Three uninfected mice of each strain were euthanased on each day and these results have been combined (n = 6). aDenotes where the level of IFN-c is significantly different from that seen in BALB/c mice (P < 0.05, protected Students t-test coupled to a one-way ANOVA). Similar results were seen in two repeat experiments. KO, knockout.
levels of TNF increased (Fig. 4D). There was no statistically significant difference in levels of IL-1b, MCP-1 or TNF between infected C57Bl/6J or P2X7R/ mice on days 8 or 12 p.i. (Fig. 4B–D), although levels of TNF in P2X7R/ mice appeared to be somewhat higher on day 8 p.i. and lower on day 12 p.i. compared with C57Bl/6J mice. Levels of IL-1b, MCP-1 and TNF were all significantly lower in infected BALB/c mice compared with C57Bl/6J and P2X7R/ mice (Fig. 4).
3.5. Production of nitric oxide is elevated in mice lacking P2X7 receptor function Classical activation of macrophages culminates in the production of pro-inflammatory mediators such as nitric oxide. This process is tightly controlled to prevent excessive immunopathology. Therefore, adhered peritoneal exudate cells from the site of infection were cultured ex vivo for 48 h and the supernatants assayed for levels of nitrite as an indicator of nitric oxide production (Fig. 5). Low levels of nitrate were detected in supernatants from adhered peritoneal exudate cells from infected BALB/c mice on days 8 and 12 p.i. Levels detected in supernatants from adhered peritoneal exudate cells from infected C57Bl/6J mice were significantly higher than those from BALB/c mice (P < 0.05) on both days
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Fig. 4. The pro-inflammatory cytokine response to Toxoplasma gondii ME49 strain is normal in mice lacking P2X7 receptor (P2X7R) function. BALB/c, C57Bl/6J and P2X7R/ mice were infected i.p. with 500 tachyzoites of T. gondii ME49, euthanased on days 8 and 12 p.i. and serum collected via cardiac puncture. Levels of IL-12 (A) and IL-1b (B) were assayed by cytokine ELISA and levels of monocyte chemoattractant protein-1(MCP-1) (C) and TNF (D) were measured by cytometric bead array as described in the Materials and methods. Results are presented as mean ± standard error of one experiment where n = 6 infected mice per strain. Three uninfected mice of each strain were euthanased on each day and these results have been combined (n = 6). aDenotes where the level of cytokine is significantly different from that seen in BALB/c mice (P < 0.05, protected Students t-test coupled to a one-way ANOVA). Similar results were seen in two repeat experiments. KO, knockout.
8 and 12 p.i. Nitrite levels in supernatants from adhered peritoneal exudate cells from P2X7R/ mice were significantly higher than BALB/c mice on both days 8 and 12 p.i. and were significantly higher than levels detected in C57Bl/6J mice on day 12 p.i. A similar pattern of nitrate production was seen in splenocytes cultured ex vivo, indicating a systemic over-production of nitric oxide in P2X7R/ mice (data not shown). 3.6. Production of IL-10 is delayed in mice lacking P2X7 receptor function
Fig. 5. Production of nitric oxide is elevated in mice lacking P2X7 receptor (P2X7R) function. BALB/c, C57Bl/6J and P2X7R/ mice were infected i.p. with 500 tachyzoites of Toxoplasma gondii ME49 strain, euthanased on days 8 and 12 p.i. and peritoneal exudate cells recovered and cultured for 48 h. Levels of nitrite in the supernatant were assayed using the Griess assay. Results are presented as mean ± standard error of one experiment where n = 6 infected mice per strain. Three uninfected mice of each strain were euthanased on each day and these results have been combined (n = 6). aDenotes that the level of nitric oxide in C57Bl/6J and P2X7R/ mice is significantly different from that seen in BALB/c mice; bdenotes that the level of nitric oxide in P2X7R/ mice is significantly different from that in C57Bl/6J mice (P < 0.05, protected Students t-test coupled to a one-way ANOVA). Similar results were seen in two repeat experiments. KO, knockout.
Signals from classically activated macrophages themselves, or surrounding cells, down-regulate macrophage activation and result in the production of anti-inflammatory cytokines such as IL10 (Mosser, 2003). This anti-inflammatory response was also altered in T. gondii-infected P2X7R/ mice. Serum IL-10 levels on day 8 p.i in infected BALB/c mice were significantly higher (P < 0.05) than levels in C57Bl/6J and P2X7R/ mice but, by day 12 p.i., levels detected in C57Bl/6J mice were significantly higher (P < 0.05) than levels in BALB/c and P2X7R/ mice (Fig. 6). 4. Discussion In this study we investigated the role of the P2X7R in the innate immune response to T. gondii by establishing a murine model using mice lacking a functional P2X7R (P2X7R/). When P2X7R/ mice
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Fig. 6. Production of IL-10 is delayed in mice lacking P2X7 receptor (P2X7R) function. BALB/c, C57Bl/6J and P2X7R/ mice were infected i.p. with 500 tachyzoites of Toxoplasma gondii ME49 strain, euthanased on days 8 and 12 p.i. and serum collected via cardiac puncture. IL-10 levels were assayed by ELISA. Results are presented as mean ± standard error of one experiment where n = 6 infected mice per strain. Three uninfected mice of each strain were euthanased on each day and these results have been combined (n = 6). aDenotes that the level of IL10 in C57Bl/6J and P2X7R/ mice is significantly different from that seen in BALB/c mice; bDenotes that the level of IL-10 in P2X7R/ mice is significantly different from that in C57Bl/6J mice (P < 0.05, protected Students t-test coupled to a one-way ANOVA). Similar results were seen in a repeat experiment. KO, knockout.
were infected with T. gondii ME49 strain and the course of acute infection monitored, they lost weight and succumbed to infection more quickly than either C57Bl/6J or BALB/c mice (Fig. 1); indeed, the differences in weight loss and mortality extant between P2X7R/ mice and C57Bl/6J were at least as large as the differences apparent between C57Bl/6J mice and BALB/c mice, a well documented susceptible versus resistant pairing (Araujo et al., 1976; Johnson, 1984; McLeod et al., 1989; Suzuki et al., 2000). To investigate the underlying cause of this susceptibility, mice of all three strains were euthanased on days 8 and 12 p.i. and signs of pathology in the liver assessed both macroscopically and microscopically (Fig. 2). Liver pathology – including inflammation and necrosis – was very evident in livers from both C57Bl/6J and P2X7R/ mice but virtually absent in BALB/c mice. This pathology is perhaps not surprising given the higher parasite burdens in the livers of the susceptible strains; the parasites no doubt attract inflammatory cells into the liver and their activation could conceivably result in extensive cell lysis and necrosis. However, the increased susceptibility of P2X7R/ mice compared with the parental C57Bl/6J mice cannot be explained on the basis of parasite burden or gross pathology. Thus, we examined production of proinflammatory cytokines since their over-production is known to contribute to immunopathology (reviewed by Miller et al., 2009a). The cytokines IL-12 and IFN-c are crucial to the development of resistance to T. gondii and have also been associated with immunopathology, so production of these two cytokines in response to infection was measured in serum from infected and uninfected mice of all three strains (Figs. 3 and 4A). Although there were slight differences in the levels of the cytokines between the three strains of mice, the same trend of levels peaking at day 8 p.i. and declining by day 12 p.i. was observed. This indicates that this part of the immune response is functioning normally despite the reduction in P2X7R function. Production of pro-inflammatory cytokines by immune effector cells such as classically activated macrophages, which can kill the parasite by oxidative and non-oxidative mechanisms, most especially via nitric oxide production, is important for control of T. gondii infection; thus, depletion of these cytokines can result in increased susceptibility to infection (reviewed by Miller et al. (2009a)). Levels of IL-1b, MCP-1 and TNF were higher in C57Bl/6J
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and P2X7R/ mice compared with BALB/c mice but there was no significant difference in levels of IL-1b, MCP-1 and TNF between C57Bl/6J and P2X7R/ mice (Fig. 4). This suggests that the high levels seen in these mice relate more to the inherent genetic differences between mice on a C57Bl/6J versus a BALB/c background and not to the activity (or lack thereof) of the P2X7R. These results are at odds with other studies showing that the P2X7R does influence production of pro-inflammatory cytokines (Ferrari et al., 1997, 1999; Sluyter et al., 2004a, b; Hewinson et al., 2008) but this result probably reflects the fact that such responses are induced by T. gondii via a number of different pathways (reviewed by Miller et al., 2009a). However, levels of nitric oxide secreted from adhered peritoneal exudate cells were significantly higher in P2X7R/ mice compared with both C57Bl/6J and BALB/c (Fig. 5). This suggests a role for the P2X7 receptor in the control of nitric oxide production. It is therefore possible, given the well established association between over-production of nitric oxide and immunopathology (Brunet, 2001), including in response to T. gondii (reviewed by Miller et al., 2009a), that the extended production of nitric oxide seen in P2X7R/ mice has contributed to their increased susceptibility. However, experiments using nitric oxide inhibitors to rescue the P2X7R/ mice from acute toxoplasmosis are needed before this conclusion can be made definitively. The cytokine IL-10 is known to play a key role in down-regulating the potent inflammatory response that develops to T. gondii, thereby helping to prevent immunopathology; thus, IL-10/ mice are unable to control the inflammatory response and succumb to infection with T. gondii due to overwhelming pathology (Gazzinelli et al., 1996; Suzuki et al., 2000). IL-10 production was initiated in all three strains of mice examined here but levels of IL-10 in infected BALB/c mice on day 8 p.i. were significantly higher than levels detected in C57Bl/6J and P2X7R/ mice (Fig. 6). By day 12 p.i., levels of IL-10 in BALB/c mice were falling, whereas they were rising in C57Bl/6J mice. Moreover, levels of IL-10 were significantly lower in P2X7R/ mice than in C57Bl/6J mice on day 12 p.i. and, in fact, had not risen significantly above levels seen on day 8 p.i. This delay in IL-10 production could conceivably contribute to the prolonged and enhanced production of nitric oxide seen in C57Bl/6J and, most particularly, P2X7R/ mice, and help explain their relative susceptibilities to T. gondii infection. This association of the P2X7R and IL-10 production is, as far as we are aware, unprecedented and therefore requires further investigation. However, our results indicate that the IL-10 response is perhaps partially, rather than exclusively, related to the activity of the P2X7R. In summary, ATP is produced as an endogenous ‘danger signal’ as a result of cellular damage inflicted by intracellular pathogens such as T. gondii or by factors such as nitric oxide during the inflammatory response. Since nitric oxide is such a toxic molecule, production is generally tightly controlled, possibly through a negative feedback mechanism whereby increased levels of nitric oxide, or danger signals (such as extracellular ATP) produced as a result of damage inflicted by nitric oxide, suppress production of inflammatory cytokines (Brunet, 2001). Our data indicate that cells lacking the P2X7R, which would be unresponsive to extracellular ATP, continue producing nitric oxide whereas cells with functional receptors are able to regulate inflammation and control nitric oxide production. Acknowledgements This work was supported by an Australian Research Council Discovery Project Grant (DP0666515) to N.C.S. and J.S.W. References Adriouch, S., Dox, C., Welge, V., Seman, M., Koch-Noble, F., Haag, F., 2002. A natural P451L mutation in the cytoplasmic domain impairs the function of the mouse P2X7 receptor. J. Immunol. 169, 4108–4112.
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International Journal for Parasitology journal homepage: www.elsevier.com/locate/ijpara
Dysregulation of the inflammatory response to the parasite, Toxoplasma gondii, in P2X7 receptor-deficient mice Catherine M. Miller a, Alana M. Zakrzewski a, Rowan J. Ikin a, Nicola R. Boulter a, Marilyn Katrib a, Michael P. Lees a, Stephen J. Fuller b, James S. Wiley b, Nicholas C. Smith a,⇑ a b
Institute for the Biotechnology of Infectious Diseases, University of Technology, Sydney, Broadway, NSW 2007, Australia Sydney Medical School, Nepean Campus, Nepean Hospital, The University of Sydney, Penrith, NSW 2751, Australia
a r t i c l e
i n f o
Article history: Received 26 September 2010 Accepted 4 October 2010 Available online 31 October 2010 Keywords: Toxoplasma gondii Apicomplexa P2X7 receptor Nitric oxide IL-10 Immunopathology
a b s t r a c t The P2X7 receptor (P2X7R) is a two transmembrane receptor that is highly expressed on the surface of immune cells. Loss of function polymorphisms in this receptor have been linked to increased susceptibility to intracellular pathogens. P2X7R gene knockout (P2X7R/; on a C57Bl/6J background), C57Bl/6J and BALB/c mice were infected with the avirulent ME49 strain of the intracellular parasite, Toxoplasma gondii, and susceptibility determined by monitoring weight loss. P2X7R/ mice lost significantly more weight than C57Bl/6J mice from day 8 p.i. C57Bl/6J, in turn, lost significantly more weight than BALB/c mice. Thus, by day 10 p.i., P2X7R/ mice had lost 5.7 ± 0.7% of their weight versus 2.4 ± 0.6% for C57Bl/6J mice, whereas BALB/c mice had gained 1.9 ± 0.5%; by day 12 p.i., P2X7R/ mice had lost 15.1 ± 0.6%, C57Bl/6J had lost 10.1 ± 0.8% and BALB/c had lost 4.8 ± 0.8% of their weight. Neither parasite burden nor liver pathology was greater in the P2X7R/ mice than in C57Bl/6J mice but BALB/c mice had significantly smaller numbers of parasites and less pathology in their livers than these strains. Absence of the P2X7 receptor did not affect IFN-c, IL-12, IL-1b, monocyte chemoattractant protein-1 (MCP-1) or TNF production. However, both P2X7R/ and C57Bl/6J mice produced more IL-1b and TNF than BALB/c mice. There was one important point of differentiation between the P2X7R/ and C57Bl/6J mice, namely the significantly enhanced and prolonged production of nitric oxide, accompanied by delayed production of IL-10 in the P2X7R-deficient mice. Ó 2010 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction Toxoplasma gondii is a protozoan parasite that is able to infect and survive in cells of the monocyte/macrophage lineage; it possesses a range of sophisticated immune evasion strategies (Montoya and Liesenfeld, 2004; Aliberti, 2005; Miller et al., 2009a). It is prevalent in humans and animals worldwide with human infection rates varying from 10% to 90% depending on factors such as animal exposure and dietary habits (Montoya and Liesenfeld, 2004). Overall, it infects approximately one-third of the world’s population and can cause serious disease in pregnant women and immuno-compromised hosts. Pathology is very rare in healthy immuno-competent people, although there are significant numbers of cases of lymphadenopathy or ocular problems around the world every year (Montoya and Liesenfeld, 2004). Acute infection is characterised by the proliferation of rapidly dividing tachyzoites ⇑ Corresponding author. Address: Institute for the Biotechnology of Infectious Diseases, University of Technology, Sydney, P.O. Box 123, Broadway, NSW 2007, Australia. Tel.: +61 2 9514 4013; fax: +61 2 9514 4201. E-mail address: [email protected] (N.C. Smith).
that stimulate production of high levels of IL-12 and IFN-c by the cells of the innate immune system. Both cytokines play a central role in the development of resistance to T. gondii. Macrophages, together with dendritic cells and neutrophils, are an important source of IL-12 early in infection (Aliberti, 2005; Miller et al., 2009a). IL-12 activates natural killer (NK) cells and, later, T cells to produce IFN-c, which in turn promotes classical activation of macrophages via a positive feedback loop (Miller et al., 2009a). Classically activated macrophages play a key role in the control of intracellular parasite growth through the production of inflammatory cytokines and nitric oxide, and trigger apoptosis of the infected cell and stage conversion of the parasite from tachyzoite to bradyzoite (Robben et al., 2004; Aliberti, 2005). However, an uncontrolled response leads to severe immunopathology (Miller et al., 2009a). Signals from surrounding cells or the macrophages themselves down-regulate macrophage activation and lead to the production of anti-inflammatory cytokines such as IL-10 and transforming growth factor-b (TGF-b). This regulation is central to controlling infection, as knockout mice lacking either of these two cytokines are more susceptible to inflammatory pathologies than wild type mice (Mosser, 2003); IL-10 plays a particularly
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significant role in preventing immunopathology in T. gondii infection (Gazzinelli et al., 1996; Suzuki et al., 2000). Recently, another factor has emerged as an important component of the inflammatory response to intracellular pathogens, namely the P2X7 receptor (P2X7R). Activation of monocyte and macrophage P2X7R has been shown to kill intracellular Mycobacterium, Chlamydia and Leishmania spp. (Molloy et al., 1994; Lammas et al., 1997; Kusner and Adams, 2000; Coutinho-Silva et al., 2001, 2003; Fairbairn et al., 2001; Stober et al., 2001; Placido et al., 2006; Darville et al., 2007; Chavez et al., 2009). The P2X7R gene is highly polymorphic and loss-of-function polymorphisms reduce control of Mycobacterium tuberculosis. Furthermore, inheritance of the 1513A>C single nucleotide polymorphism has been associated with susceptibility to extrapulmonary tuberculosis in humans (Saunders et al., 2003; Fernando et al., 2005, 2007; Shemon et al., 2006; Nino-Moreno et al., 2007). The P2X7R is an ATP-gated cation channel belonging to the P2X family of receptors. It is widely expressed by cells of the immune system (Chen and Brosnan, 2006). Brief exposure to ATP leads to an influx of Ca2+ and Na+ and an efflux of K+, while prolonged or repeated exposure causes the formation of large pores in the membrane of the cell that allows the passage of molecules up to 314 Da in size (Wiley et al., 2001). The main source of the ATP is extracellular ATP (eATP) released from activated platelets, endothelial cells, nerve cells and antigen-stimulated T cells, and from stressed or dying cells during an inflammatory response (Aga et al., 2004; Tsukimoto et al., 2006). Stimulation of the P2X7R is regarded as a key process in the inflammatory response to infection as it potentiates the release of various pro-inflammatory mediators such as IL-1b, TNF and nitric oxide, activates phospholipase D leading to phagosome/lysosome fusion, and induces cell death through apoptosis and/or necrosis (Coutinho-Silva et al., 2007; Lenertz et al., 2009). It has been reported recently that activation of the P2X7R by ATP can mediate killing of T. gondii (Correa et al., 2010; Lees et al., 2010) via, potentially, either apoptosis (Lees et al., 2010) or formation of phagolysosomes accompanied by generation of reactive oxygen species (Correa et al., 2010). These observations, coupled with the fact that many pathways involved with P2X7R activation are also typical of innate Th1 responses, makes it conceivable that the P2X7R has an important role in the induction of the immune response to T. gondii. In this study we demonstrate that mice lacking a P2X7 receptor (P2X7R/) are more susceptible to T. gondii infection than mice with half-functional and fully functional P2X7 receptors and this is associated with an apparent lack of regulation of the inflammatory response.
2. Materials and methods 2.1. Mice All animal research was performed with the approval of the University of Technology Sydney (UTS; Australia)/Royal North Shore Hospital Animal Care & Ethics Committee Protocols UTS/ RNSH 0611-042A and UTS ACEC 2008-03. Pathogen-free, 6–8 week old C57Bl/6J and BALB/c mice were obtained from the Gore Hill Research Laboratories (Sydney, Australia) or Animal Resource Centre (Perth, Western Australia). P2RX7 gene-deleted mice (P2X7R/) on a C57Bl/6J background (back-crossed for at least seven generations) were originally provided by Pfizer, Inc. (Ann Arbor, MI, USA) and were subsequently bred at the Gore Hill Research Laboratories and the Ernst Facility (UTS, Australia). C57BL/6 mice have a proline to leucine polymorphism at amino acid 451 in the C-terminal tail of the P2X7R that has various effects on P2X7R function (Adriouch et al., 2002; Le Stunff et al., 2004; Asward and Dennert, 2006; Hong et al., 2009; Suadicani et al., 2009; Yip et al., 2009).
Thus, we also included comparative analyses of BALB/c cells in our investigations, as this strain has fully functional P2X7R (Adriouch et al., 2002; Le Stunff et al., 2004; Asward and Dennert, 2006; Hong et al., 2009; Suadicani et al., 2009; Yip et al., 2009) and is also known to be more resistant to T. gondii than C57BL/6J mice (Araujo et al., 1976; Johnson, 1984; McLeod et al., 1989; Suzuki et al., 2000). Inclusion of the BALB/c infections in our analyses was important to internally define a resistant phenotype; thus, we were looking for differences between P2X7R/ mice and C57Bl/6J mice that were comparable to differences observed between C57Bl/6J mice and BALB/c mice, a recognised susceptible versus resistant pairing (Araujo et al., 1976; Johnson, 1984; McLeod et al., 1989; Suzuki et al., 2000). Lack of the P2RX7 gene in the knockout mice was confirmed by PCR using the following primers: For 50 -CTATCTCTCCACGACT CACCCCC-30 and Rev 50 -TATAATCCCGGGAGGGA TACTTGAAGCCAC TGTAC-30 (Adriouch et al., 2002). Lack of P2X7R function in splenocytes from uninfected P2X7R/ mice was also confirmed by measuring ATP-induced ethidium uptake as described previously (Lees et al., 2010). 2.2. Parasites and infections Tachyzoites of the T. gondii ME49 strain were obtained from the American Type Culture Collection (Manassas, VA, USA) and subsequently maintained by continuous passage in Vero cells (using RPMI from Invitrogen and 2% heat inactivated newborn calf serum) at 37 °C in a 5% CO2 incubator. Parasites were harvested from freshly lysed cultures, passed through a 26 G needle and concentrated by spinning at 500g. The pellet of tachyzoites was resuspended in sterile 0.9% saline. Parasites were counted using a Neubauer haemocytometer and diluted in sterile 0.9% saline to the required dose for injection. i.p. injection of tachyzoites was favoured for our model rather than oral infection with brain cysts because estimation of the infective dose of tachyzoites is more reliable than that of bradyzoites since each tissue cyst contains varying numbers of bradyzoites. Furthermore, the dissemination patterns following oral infection of mice with tissue cysts can be inconsistent (Boyle et al., 2007) and, ultimately, both infection routes result in similar immune responses, with i.p. infection leading to faster, more reproducible effects (Fouts and Boothroyd, 2007). To assess relative susceptibility to T. gondii infection, male mice of each strain were infected i.p. with 500 tachyzoites of T. gondii ME49 and monitored daily for clinical signs of T. gondii infection – weight loss, ruffled fur, hunched posture, lethargy and morbidity – for up to 3 weeks. Mice that showed signs of morbidity or had lost >20% of starting weight were euthanased by terminal anaesthesia as per Animal Care & Ethics Protocol stipulations. In total, 27 P2X7R/ mice, 27 C57Bl/6J mice and 28 BALB/c mice were infected over the course of three separate experiments. A total of 14, 13 and 12 uninfected P2X7R/, C57Bl/6J and BALB/c mice, respectively, served as uninfected controls. To identify aspects of the innate immune response that may be implicated in the increased susceptibility of P2X7R/ mice to T. gondii infection, mice were also euthanased at set time points. Two experiments were conducted where six mice of each strain were infected i.p. with 500 T. gondii ME49 tachyzoites and three from each strain were euthanased on days 8 and 12 p.i. In a third experiment, 12 mice from each strain were infected i.p. with 500 T. gondii ME49 tachyzoites and six from each strain were euthanased on days 8 and 12 p.i. 2.3. Assessment of parasite burden and liver pathology Relative parasite burdens were assessed in liver sections using immunohistology with a rabbit anti-T. gondii polyclonal antibody
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(RayBiotech, Inc., Norcross, GA, USA). Unstained formalin-fixed, paraffin-embedded sections of liver (4 lm thick) were de-paraffinised and antigen retrieval performed by heating in 10 mM citrate buffer pH 6.5. Immunoperoxidase staining was then performed using anti-rabbit secondary antibodies in an ABC staining system (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) according to the manufacturer’s instructions. Controls included incubating with no primary antibody or an unrelated rabbit IgG. Slides were dehydrated, mounted and examined using a light microscope and the number of parasitophorous vacuoles containing tachyzoites or tachyzoite antigen counted in five fields of view under 10 objective by two independent investigators. The livers of all animals were removed and assessed visually for signs of necrosis. Livers were scored according to the following criteria: (0) normal colour, no visible necrosis; (1) slight change in colour, no visible necrosis; (2) change in colour with moderate lesions; (3) change in colour with severe necrosis. Sections of formalin-fixed, paraffin-embedded livers (4 lm thick) from two infected and one uninfected mouse per strain on each day of infection were also examined for areas of necrosis and inflammation following staining with H & E. Sections were scored according to the following criteria. Cellular infiltrate: (0) absent; (1) one focal infiltrate; (2) multifocal infiltrates (<10 per field); 3-diffuse infiltrate (>10 per field). Necrosis: (0) no visible necrosis; (1) small areas of necrosis; (2) moderate levels of necrosis (<50%); (3) marked necrosis (>50%). Five fields of view using the 10 objective were scored per section by two independent investigators blinded to the strain of mouse and infection status. 2.4. Isolation and ex vivo culture of peritoneal exudate cells Peritoneal exudate cells were recovered by rinsing the peritoneal cavity with 6 ml of sterile PBS, pelleted at 500g, then resuspended in RPMI 1640 containing 10% newborn calf serum and cultured in six well plates at 37 °C/5% CO2. After 2–3 h incubation, non-adhered cells were removed by washing with RPMI and adhered cells (previously determined to be 89–91% macrophages; Miller et al., 2009b) were removed by scraping. Cells were counted, adjusted to a concentration of 1 106/ml and replated into 48 well plates. Cells were incubated at 37 °C/5% CO2 for 48 h before supernatants were removed and stored at 20 °C until analysis. Supernatants were assayed for nitric oxide as described below.
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Nitric oxide concentrations were determined using the Griess assay as described previously (Miller et al., 2000). Briefly, nitric oxide produced by adhered peritoneal exudate cells was measured by mixing equal amounts of supernatant with Griess reagent (1% sulfanilimide in 2.5% H3PO4 and 0.1% napthylethylenediamine dihydrochloride in 2.5% H3PO4 mixed in a 1:1 ratio just prior to assay). Absorbance was measured at 540 nm and the amount of nitrite present was determined by comparing against a standard curve generated with known concentrations of sodium nitrite. 2.6. Statistical analyses The statistical significance of differences between groups was determined using a protected Students t-test coupled to a oneway ANOVA. A P value of <0.05 was considered significant. 3. Results 3.1. P2X7R/ mice are more susceptible to acute T. gondii infection than C57Bl/6J or BALB/c mice Susceptibility of P2X7R/ mice to acute T. gondii infection was assessed relative to susceptibility of C57Bl/6J mice and BALB/c mice by infecting mice with T. gondii ME49 strain and monitoring the course of infection. Mice were euthanased when they showed signs of morbidity or had lost >20% of starting weight. All infected mice exhibited clinical signs of infection and lost weight relative to uninfected mice of the same strain (Fig. 1). P2X7R/ mice lost significantly more weight than C57Bl/6J, which lost significantly more weight than BALB/c mice, beginning on day 8 p.i. Thus, by day 10 p.i., P2X7R/ mice had lost 5.7 ± 0.7%of their body weight versus 2.4 ± 0.6% for C57Bl/6J mice, whereas BALB/c mice had gained 1.9 ± 0.5%; by day 12 p.i., P2X7R/ mice had lost 15.1 ± 0.6%, C57Bl/6J had lost 10.1 ± 0.8% and BALB/c had lost 4.8 ± 0.8% of their body weight. Moreover, P2X7R/ mice succumbed to infection earlier so that, by day 18 p.i., 52% of the knockout mice had to be euthanased, whereas only 26% of the C57BL/6J and none of the BALB/c mice had to be euthanased. By day 21 p.i., 74% of the P2X7R/ mice had to be euthanased compared with 37% of C57Bl/6J and 36% of BALB/c mice.
2.5. Immunological assays Splenocytes were cultured at 5 106 cells/ml for 72 h without stimulation or with 50 lg/ml of T. gondii soluble extract or anticlusters of differentiation 3 (CD3; 2 lg/ml) plus phorbol 12-myristate 13 acetate (PMA) (25 ng/ml). Concentrations of the cytokines IL-12, IFN-c, IL-1b and IL-10 in serum and splenocyte medium were measured by immunoassay using matched pairs of anti-cytokine antibodies purchased from BD Pharmingen (San Diego, CA, USA) according to the manufacturer’s instructions. Recombinant cytokines of known concentration were used to generate standard curves. Levels of the pro-inflammatory cytokines, monocyte chemoattractant protein-1 (MCP-1) and TNF were measured using a BD Cytometric Bead Array kit (San Diego, CA, USA) according to the manufacturer’s instructions. Briefly, the serum samples and test standards supplied were mixed with cytokine capture beads and phycoerythrin (PE)-conjugated antibodies, and incubated for 2 h in the dark. After washing away unbound sample and reagent, fluorescence was measured in individual standards and samples on a BD LSR-II flow cytometer. Following data acquisition, standard curves were generated and cytokine amounts quantified using FCAP Array, version 1.0.1 (BD, San Diego, CA, USA).
Fig. 1. Weight loss caused by infection with Toxoplasma gondii is exacerbated in mice lacking P2X7 receptor (P2X7R) function. BALB/c, C57Bl/6J and P2X7R/ mice were infected i.p. with 500 tachyzoites of T. gondii ME49 strain and weighed daily. Cumulative weight loss of P2X7R/ mice was significantly greater than that of C57Bl/6J mice which, in turn, was significantly greater than that of BALB/c mice on each of days 8–14 p.i. (P < 0.05, protected Students t-test coupled to a one-way ANOVA; n = 27 for infected P2X7R/ and C57Bl/6J mice and n = 28 for BALB/c mice; n = 14, 13 and 12 for uninfected P2X7R/, C57Bl/6J and BALB/c mice, respectively). B-U = uninfected BALB/c, B-I = infected BALB/c, C-U = uninfected C57Bl/6J, CI = infected C57Bl/6J, P-U = uninfected P2X7R/ and P-I = infected P2X7R/ mice.
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Fig. 2. Parasite burden, gross liver pathology and histopathology in Toxoplasma gondii ME49-infected mice are not affected by the absence of the P2X7 receptor (P2X7R). Parasite burden in livers of BALB/c, C57Bl/6J and P2X7R/ mice (A) was determined by immunohistochemistry as detailed in the Materials and methods. Gross liver pathology (B) was assessed visually by scoring between 0 and 3 with a score of 0 indicating no visible necrosis and 3 indicating severe necrosis. Histopathology was assessed by examining H & E stained slides for areas of inflammation and necrosis ((C–E) pictures shown are from single mice and are representative of all mice examined in the groupfilled arrows indicate areas of cellular infiltrate and unfilled arrows indicate areas of necrosis). Levels of cellular infiltrate (G) and necrosis (H) were scored; a score of 0 indicating no inflammation or necrosis was evident while a score of 3 indicates high levels of infiltrate or necrosis. Scores are presented as mean ± standard error, n = 6 for gross liver pathology, n = 2 for histopathology and parasite burden. aIndicates where the score is significantly different from the score for BALB/c mice (P < 0.05, protected Students t-test coupled to a one-way ANOVA). KO, knockout.
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3.2. P2X7R/ and C57Bl/6J mice exhibit higher parasite burdens and extended liver pathology compared with BALB/c mice On days 8 and 12 p.i., parasite burden and liver pathology were assessed (Fig. 2). Cells containing T. gondii tachyzoites or antigen in liver sections were identified with an anti-T. gondii antibody and immunoperoxidase staining. Parasite burden and liver pathology were essentially identical in P2X7R/ and C57Bl/6J mice (Fig. 2A-H). However, significantly fewer areas containing parasites were identified in sections from BALB/c mice than in either C57Bl/ 6J mice or P2X7R/ mice on day 12 p.i. (Fig. 2A). Likewise, less gross pathology was evident in livers from BALB/c mice on both days 8 and 12 p.i., although the differences were more pronounced on day 12 p.i. There was little change in the scores for BALB/c mice on day 12 p.i. compared with day 8 p.i. but both C57Bl/6J and P2X7R/ mice showed a marked increase in pathology from day 8 p.i. to day 12 p.i. (Fig. 2B). Microscopic analysis of H & E stained sections from all three strains of mice confirmed these observations. Levels of cellular infiltrate and necrosis were similar in all three strains of mice on day 8 p.i. but, again, differences were quite pronounced on day 12 p.i. (Fig. 2C–G). Sections from infected P2X7R/ and C57Bl/6J mice (Fig. 2E–G) showed higher levels of infiltrate and larger areas of necrosis (Fig. 2E, F, and H) compared with sections from BALB/c mice (Fig. 2D, G, and H). No areas of cellular infiltrate or necrosis were seen in sections from uninfected mice (Fig. 2C, G, and H). 3.3. The IFN-c response to T. gondii infection is normal in mice with reduced or no P2X7R function IFN-c is crucial for resistance to T. gondii infection. Therefore, levels of this cytokine were assessed in serum from all three strains of mice on days 8 and 12 p.i. Levels of IFN-c were significantly elevated (P < 0.05) in infected mice of all three strains compared with uninfected mice of the same strain on both experimental days (Fig. 3A). All three strains of mice also showed a similar pattern of IFN-c production with a peak on day 8 p.i. followed by a decline in levels on day 12 p.i., although there were slight differences in amounts produced between the three strains (Fig. 3A). Levels of IFN-c were also assessed in supernatants from splenocytes cultured without antigenic stimulation (Fig. 3B). Again, a similar pattern of IFN-c production was seen amongst the three strains of mice with a peak on day 8 p.i. followed by a decline on day 12 p.i. There was no significant difference in IFN-c levels amongst the three strains of mice, either in serum (Fig. 3A) or splenocytes, whether stimulated with T. gondii antigen or PMA/anti-CD3 (data not shown) or not stimulated (Fig. 3B). 3.4. Production of pro-inflammatory cytokines is unaltered in mice lacking P2X7 receptor function compared with wild type mice Classical activation of macrophages results in the production of pro-inflammatory cytokines such as IL-12, IL-1b, MCP-1 and TNF. Levels of these cytokines were measured in the serum of infected mice from all three strains on days 8 and 12 p.i. (Fig. 4). Levels of IL-12 (Fig. 4A) were significantly elevated (P < 0.05) in infected mice of all three strains compared with uninfected mice of the same strain on both experimental days (Fig. 4A). All three strains of mice also showed a similar pattern of IL-12 production, with a peak on day 8 p.i. followed by a decline in levels on day 12 p.i., although there were slight differences in amounts produced between the three strains (Fig. 4A). There were significant increases (P < 0.05) in the levels of IL-1b (Fig. 4B), MCP-1 (Fig. 4C) and TNF (Fig. 4D) compared with uninfected mice on day 8 p.i. in C57Bl/ 6J and P2X7R/ mice and, subsequently, levels of IL-1b (Fig. 4B) and MCP-1 (Fig. 4C) declined significantly by day 12 p.i. whereas
Fig. 3. The IFN-c response to Toxoplasma gondii ME49 strain is normal in mice lacking P2X7 receptor (P2X7R) function. BALB/c, C57Bl/6J and P2X7R/ mice were infected i.p. with 500 tachyzoites of T. gondii ME49, euthanased on days 8 and 12 p.i. and serum collected via cardiac puncture. Splenocytes were cultured in vitro for 3 days before the supernatant was collected. Levels of IFN-c in serum (A) and spleen cell supernatant (B) were assayed by ELISA. Results are presented as mean ± standard error of one experiment where n = 6 infected mice per strain. Three uninfected mice of each strain were euthanased on each day and these results have been combined (n = 6). aDenotes where the level of IFN-c is significantly different from that seen in BALB/c mice (P < 0.05, protected Students t-test coupled to a one-way ANOVA). Similar results were seen in two repeat experiments. KO, knockout.
levels of TNF increased (Fig. 4D). There was no statistically significant difference in levels of IL-1b, MCP-1 or TNF between infected C57Bl/6J or P2X7R/ mice on days 8 or 12 p.i. (Fig. 4B–D), although levels of TNF in P2X7R/ mice appeared to be somewhat higher on day 8 p.i. and lower on day 12 p.i. compared with C57Bl/6J mice. Levels of IL-1b, MCP-1 and TNF were all significantly lower in infected BALB/c mice compared with C57Bl/6J and P2X7R/ mice (Fig. 4).
3.5. Production of nitric oxide is elevated in mice lacking P2X7 receptor function Classical activation of macrophages culminates in the production of pro-inflammatory mediators such as nitric oxide. This process is tightly controlled to prevent excessive immunopathology. Therefore, adhered peritoneal exudate cells from the site of infection were cultured ex vivo for 48 h and the supernatants assayed for levels of nitrite as an indicator of nitric oxide production (Fig. 5). Low levels of nitrate were detected in supernatants from adhered peritoneal exudate cells from infected BALB/c mice on days 8 and 12 p.i. Levels detected in supernatants from adhered peritoneal exudate cells from infected C57Bl/6J mice were significantly higher than those from BALB/c mice (P < 0.05) on both days
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Fig. 4. The pro-inflammatory cytokine response to Toxoplasma gondii ME49 strain is normal in mice lacking P2X7 receptor (P2X7R) function. BALB/c, C57Bl/6J and P2X7R/ mice were infected i.p. with 500 tachyzoites of T. gondii ME49, euthanased on days 8 and 12 p.i. and serum collected via cardiac puncture. Levels of IL-12 (A) and IL-1b (B) were assayed by cytokine ELISA and levels of monocyte chemoattractant protein-1(MCP-1) (C) and TNF (D) were measured by cytometric bead array as described in the Materials and methods. Results are presented as mean ± standard error of one experiment where n = 6 infected mice per strain. Three uninfected mice of each strain were euthanased on each day and these results have been combined (n = 6). aDenotes where the level of cytokine is significantly different from that seen in BALB/c mice (P < 0.05, protected Students t-test coupled to a one-way ANOVA). Similar results were seen in two repeat experiments. KO, knockout.
8 and 12 p.i. Nitrite levels in supernatants from adhered peritoneal exudate cells from P2X7R/ mice were significantly higher than BALB/c mice on both days 8 and 12 p.i. and were significantly higher than levels detected in C57Bl/6J mice on day 12 p.i. A similar pattern of nitrate production was seen in splenocytes cultured ex vivo, indicating a systemic over-production of nitric oxide in P2X7R/ mice (data not shown). 3.6. Production of IL-10 is delayed in mice lacking P2X7 receptor function
Fig. 5. Production of nitric oxide is elevated in mice lacking P2X7 receptor (P2X7R) function. BALB/c, C57Bl/6J and P2X7R/ mice were infected i.p. with 500 tachyzoites of Toxoplasma gondii ME49 strain, euthanased on days 8 and 12 p.i. and peritoneal exudate cells recovered and cultured for 48 h. Levels of nitrite in the supernatant were assayed using the Griess assay. Results are presented as mean ± standard error of one experiment where n = 6 infected mice per strain. Three uninfected mice of each strain were euthanased on each day and these results have been combined (n = 6). aDenotes that the level of nitric oxide in C57Bl/6J and P2X7R/ mice is significantly different from that seen in BALB/c mice; bdenotes that the level of nitric oxide in P2X7R/ mice is significantly different from that in C57Bl/6J mice (P < 0.05, protected Students t-test coupled to a one-way ANOVA). Similar results were seen in two repeat experiments. KO, knockout.
Signals from classically activated macrophages themselves, or surrounding cells, down-regulate macrophage activation and result in the production of anti-inflammatory cytokines such as IL10 (Mosser, 2003). This anti-inflammatory response was also altered in T. gondii-infected P2X7R/ mice. Serum IL-10 levels on day 8 p.i in infected BALB/c mice were significantly higher (P < 0.05) than levels in C57Bl/6J and P2X7R/ mice but, by day 12 p.i., levels detected in C57Bl/6J mice were significantly higher (P < 0.05) than levels in BALB/c and P2X7R/ mice (Fig. 6). 4. Discussion In this study we investigated the role of the P2X7R in the innate immune response to T. gondii by establishing a murine model using mice lacking a functional P2X7R (P2X7R/). When P2X7R/ mice
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Fig. 6. Production of IL-10 is delayed in mice lacking P2X7 receptor (P2X7R) function. BALB/c, C57Bl/6J and P2X7R/ mice were infected i.p. with 500 tachyzoites of Toxoplasma gondii ME49 strain, euthanased on days 8 and 12 p.i. and serum collected via cardiac puncture. IL-10 levels were assayed by ELISA. Results are presented as mean ± standard error of one experiment where n = 6 infected mice per strain. Three uninfected mice of each strain were euthanased on each day and these results have been combined (n = 6). aDenotes that the level of IL10 in C57Bl/6J and P2X7R/ mice is significantly different from that seen in BALB/c mice; bDenotes that the level of IL-10 in P2X7R/ mice is significantly different from that in C57Bl/6J mice (P < 0.05, protected Students t-test coupled to a one-way ANOVA). Similar results were seen in a repeat experiment. KO, knockout.
were infected with T. gondii ME49 strain and the course of acute infection monitored, they lost weight and succumbed to infection more quickly than either C57Bl/6J or BALB/c mice (Fig. 1); indeed, the differences in weight loss and mortality extant between P2X7R/ mice and C57Bl/6J were at least as large as the differences apparent between C57Bl/6J mice and BALB/c mice, a well documented susceptible versus resistant pairing (Araujo et al., 1976; Johnson, 1984; McLeod et al., 1989; Suzuki et al., 2000). To investigate the underlying cause of this susceptibility, mice of all three strains were euthanased on days 8 and 12 p.i. and signs of pathology in the liver assessed both macroscopically and microscopically (Fig. 2). Liver pathology – including inflammation and necrosis – was very evident in livers from both C57Bl/6J and P2X7R/ mice but virtually absent in BALB/c mice. This pathology is perhaps not surprising given the higher parasite burdens in the livers of the susceptible strains; the parasites no doubt attract inflammatory cells into the liver and their activation could conceivably result in extensive cell lysis and necrosis. However, the increased susceptibility of P2X7R/ mice compared with the parental C57Bl/6J mice cannot be explained on the basis of parasite burden or gross pathology. Thus, we examined production of proinflammatory cytokines since their over-production is known to contribute to immunopathology (reviewed by Miller et al., 2009a). The cytokines IL-12 and IFN-c are crucial to the development of resistance to T. gondii and have also been associated with immunopathology, so production of these two cytokines in response to infection was measured in serum from infected and uninfected mice of all three strains (Figs. 3 and 4A). Although there were slight differences in the levels of the cytokines between the three strains of mice, the same trend of levels peaking at day 8 p.i. and declining by day 12 p.i. was observed. This indicates that this part of the immune response is functioning normally despite the reduction in P2X7R function. Production of pro-inflammatory cytokines by immune effector cells such as classically activated macrophages, which can kill the parasite by oxidative and non-oxidative mechanisms, most especially via nitric oxide production, is important for control of T. gondii infection; thus, depletion of these cytokines can result in increased susceptibility to infection (reviewed by Miller et al. (2009a)). Levels of IL-1b, MCP-1 and TNF were higher in C57Bl/6J
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and P2X7R/ mice compared with BALB/c mice but there was no significant difference in levels of IL-1b, MCP-1 and TNF between C57Bl/6J and P2X7R/ mice (Fig. 4). This suggests that the high levels seen in these mice relate more to the inherent genetic differences between mice on a C57Bl/6J versus a BALB/c background and not to the activity (or lack thereof) of the P2X7R. These results are at odds with other studies showing that the P2X7R does influence production of pro-inflammatory cytokines (Ferrari et al., 1997, 1999; Sluyter et al., 2004a, b; Hewinson et al., 2008) but this result probably reflects the fact that such responses are induced by T. gondii via a number of different pathways (reviewed by Miller et al., 2009a). However, levels of nitric oxide secreted from adhered peritoneal exudate cells were significantly higher in P2X7R/ mice compared with both C57Bl/6J and BALB/c (Fig. 5). This suggests a role for the P2X7 receptor in the control of nitric oxide production. It is therefore possible, given the well established association between over-production of nitric oxide and immunopathology (Brunet, 2001), including in response to T. gondii (reviewed by Miller et al., 2009a), that the extended production of nitric oxide seen in P2X7R/ mice has contributed to their increased susceptibility. However, experiments using nitric oxide inhibitors to rescue the P2X7R/ mice from acute toxoplasmosis are needed before this conclusion can be made definitively. The cytokine IL-10 is known to play a key role in down-regulating the potent inflammatory response that develops to T. gondii, thereby helping to prevent immunopathology; thus, IL-10/ mice are unable to control the inflammatory response and succumb to infection with T. gondii due to overwhelming pathology (Gazzinelli et al., 1996; Suzuki et al., 2000). IL-10 production was initiated in all three strains of mice examined here but levels of IL-10 in infected BALB/c mice on day 8 p.i. were significantly higher than levels detected in C57Bl/6J and P2X7R/ mice (Fig. 6). By day 12 p.i., levels of IL-10 in BALB/c mice were falling, whereas they were rising in C57Bl/6J mice. Moreover, levels of IL-10 were significantly lower in P2X7R/ mice than in C57Bl/6J mice on day 12 p.i. and, in fact, had not risen significantly above levels seen on day 8 p.i. This delay in IL-10 production could conceivably contribute to the prolonged and enhanced production of nitric oxide seen in C57Bl/6J and, most particularly, P2X7R/ mice, and help explain their relative susceptibilities to T. gondii infection. This association of the P2X7R and IL-10 production is, as far as we are aware, unprecedented and therefore requires further investigation. However, our results indicate that the IL-10 response is perhaps partially, rather than exclusively, related to the activity of the P2X7R. In summary, ATP is produced as an endogenous ‘danger signal’ as a result of cellular damage inflicted by intracellular pathogens such as T. gondii or by factors such as nitric oxide during the inflammatory response. Since nitric oxide is such a toxic molecule, production is generally tightly controlled, possibly through a negative feedback mechanism whereby increased levels of nitric oxide, or danger signals (such as extracellular ATP) produced as a result of damage inflicted by nitric oxide, suppress production of inflammatory cytokines (Brunet, 2001). Our data indicate that cells lacking the P2X7R, which would be unresponsive to extracellular ATP, continue producing nitric oxide whereas cells with functional receptors are able to regulate inflammation and control nitric oxide production. Acknowledgements This work was supported by an Australian Research Council Discovery Project Grant (DP0666515) to N.C.S. and J.S.W. References Adriouch, S., Dox, C., Welge, V., Seman, M., Koch-Noble, F., Haag, F., 2002. A natural P451L mutation in the cytoplasmic domain impairs the function of the mouse P2X7 receptor. J. Immunol. 169, 4108–4112.
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