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The Soluble Tachyzoite Antigen of Toxoplasma gondii Has a Protective Effect on Mouse Allografts
The Soluble Tachyzoite Antigen of Toxoplasma gondii Has a Protective Effect on Mouse Allografts S. Wang, Z. Fang, X. Huang, X. Luo, Z. Fang, N. Gong, and C. Ming ABSTRACT Background. Infection with some types of parasites can significantly prolong allograft survival in mice. It is unknown whether the soluble tachyzoite antigen (STAg) from Toxoplasma gondii has the same effect and by what mechanism it acts. Methods. A mouse model of cardiac and skin allograft transplantation was established between BALB/c (H-2d) and C57BL/6(H-2b) mice. T gondii STAg was prepared, and 5 g was administered subcutaneously to recipient mice 4 days before transplantation. The graft status was checked daily, and histologic and immunohistochemical assays were used to evaluate rejection. The serum cytokine levels from the recipient mice were analyzed by Luminex. Result. The administration of 5 g STAg 4 days before transplantation significantly prolonged the survival time of the heart and skin allografts to 85.17 ⫾ 14.06 and 24.17 ⫾ 2.32 days, respectively. Immunohistochemical staining showed that the CD4⫹ and CD8⫹ T lymphocytes were markedly reduced in the allografts at day 7 posttransplantation. Notably, interleukin (IL)-12, IL-2, and IL-17 levels were significantly reduced in the serum of mice treated with STAg compared with untreated mice 7 days after transplantation. In contrast, the levels of the antiinflammatory cytokine IL-10 were elevated. Conclusion. A single administration of STAg before transplantation can significantly prolong the allograft survival time, which is accompanied by impaired lymphocyte infiltration and a reduced Th1 response. N MODERN MEDICINE, organ transplantation has been developed to treat patients undergoing end-stage organ failure. The recipients of transplants must use nonspecific immunosuppressive drugs to prevent organ rejection. However, long-term treatment with anti-rejection drugs can increase the risk of developing malignancies and infections.1 The ultimate goal for transplantation is to avoid these complications by specifically inducing tolerance without resorting to chronic immunosuppressive therapy. Toxoplasma gondii is an intracellular protozoan parasite that infects approximately one-third of the world’s population, making T gondii a remarkably successful organism. T gondii has developed several strategies to evade the host defense system, including triggering the secretion of anti-inflammatory cytokines, such as interleukin (IL)-10 and transforming growth factor (TGF)-, and suppressing the host Th1 immune response.2– 4 Applying these evasive strategies to graft transplantation could prolong survival and serve as a new therapy to avoid systemic immunosuppression.
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© 2013 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 45, 677– 683 (2013)
We previously observed that T gondii infection before transplantation significantly prolonged the survival time of allografts in rats while simultaneously suppressing inflammation. We hypothesize that transplantation is similar to parasitism and that chronic parasite infection could prolong allograft survival.5 From the Institute of Organ Transplantation (S.W., Ze.F., X.H., X.L., N.G., C.M.), Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Key Laboratory of Ministry of Health, Key Laboratory of Ministry of Education, Wuhan, China; and Department of Parasite (Z.F.), Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China. This work was supported by the National Natural Science and Foundation of China, grant 30772052. Address correspondence to Changsheng Ming, MD, PhD, Institute of Organ Transplantation, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China. E-mail: [email protected] 0041-1345/–see front matter http://dx.doi.org/10.1016/j.transproceed.2012.02.036 677
678
OBJECTIVE
In this study, we investigated whether soluble tachyzoite antigen (STAg) from T gondii has the same protective effects as the whole parasite on mouse heart and skin allografts and explored the mechanisms that are involved in this protection. MATERIALS AND METHODS Parasites and STAg The T gondii (RH strain) was a gift from the Parasite Division of Tongji Medical School and was used to infect BALB/c (H-2d) mice. STAg was isolated from T gondii (RH strain) tachyzoites grown in infected BALB/c (H-2d) mice. In brief, the peritoneal lavage fluid of the infected mice was purified by filtration through a 5-m diameter cellulose membrane. After sonication, the samples were centrifuged at 9000 g for 30 minutes. The resulting supernatant is referred to as T gondii STAg.
Animal Treatment and Cardiac or Skin Transplantation C57BL/6 (H-2b) mice were injected subcutaneously with 5 g of STAg 4 days before cardiac or skin transplantation. The cardiac transplantation was performed as previously reported.6 Briefly, after the recipient mice were anesthetized, the cardiac grafts obtained from BALB/c (H-2d) or C57BL/6 (H-2b) mice were transplanted into the cervical position of the recipient mice, with end-to-end anastomoses between the donor heart aorta and recipient carotid artery, and the donor pulmonary artery and recipient jugular vein. The total cessation of cardiac contraction was defined as the endpoint. Full tail skin was harvested from the sacrificed BALB/c (H-2d) mice. The skin was cut to a size of 8 ⫻ 8 mm. The C57BL/6 (H-2b) mice were anesthetized and shaved around the chest and backside, and a 9 ⫻ 9 mm square of skin was removed from the backside. The skin allografts were fixed on the graft beds with adhesive plaster. The rejection was diagnosed by cyanosis, erythema, erosion, and loss of the skin graft.
WANG, FANG, HUANG ET AL incubated for 2 hours, after which the biotinylated detector antibody was added. The plates were then washed and streptavidinRPE was added. After a final wash, the plates were analyzed using a Luminex 200 instrument.
Western Blot Analysis Equal amounts of tissue protein (30 g) per lane were electrophoresed on a 12% SDS-PAGE gel for separation and then transferred to PVDF membranes. After blocking, the membranes were incubated with primary antibodies against nuclear factor (NF)-B or -actin overnight at 4°C. The membranes were then incubated with a secondary antibody (HRP-conjugated goat anti-rabbit IgG). The peroxidase activity was detected using the ECL assay and analyzed by densitometry and image software.
Quantitative Real-Time Polymerase Chain Reaction Total RNA was extracted from the heart grafts using Trizol (Invitrogen, Grand Island, NY) as per the manufacturer’s protocol. Quantitative real-time polymerase chain reactions (qRT-PCRs) were performed using Rotor Gene 3000 (Corbett Research, Sydney, Australia) with a Quantitect SYBR Green PCR Kit (Toyobo,
Histopathology and Immunohistochemistry Allograft samples for histology were processed routinely by the histology laboratory. The tissue was fixed in 10% formalin and then embedded in paraffin before the preparation of sections. The tissue sections were stained with hematoxylin and eosin (H & E). Histologic rejection was evaluated by ⱖ2 experienced pathologists blinded to the source of the experimental animals and histology tissues. Immunohistochemical staining was performed on 4-mthick sections cut from formalin-fixed, paraffin-embedded tissue. Briefly, after deparaffinization and hydration, the sections were blocked with 3% H2O2 and 10% normal goat serum. The sections were incubated with a primary antibody overnight at 4°C. After washing, the sections were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin (Ig)G for 30 minutes at 37°C, followed by visualization using DAB.
Luminex Analysis of the Cytokines in the Serum For the Luminex assay, 25 L of the diluted bead solution was added to each well, after which the beads were washed. Next, 50 L of the incubation buffer and 100 L of the appropriate standard dilution was added to the wells designated for the standard curve, and 50 L of the assay diluent followed by 50 L of the sample was added to the wells designated for the samples. The samples were
Fig 1. Survival times of the heart and skin allografts after STAg treatment. The heterotopic heart transplants from the BALB/c (H-2d) donor mice to the C57BL/6 (H-2b) recipients were monitored daily by manual palpation. The rejection of the cardiac grafts was defined as the cessation of graft pulsing. The rejection of the skin transplants from the BALB/c (H-2d) donor mice to the C57BL/6 (H-2b) recipients was defined as the presence of cyanosis, erythema, erosion, and loss of the skin graft. The recipient mice were divided into groups receiving PBS (control group, n ⫽ 6), 5 g STAg (n ⫽ 6), or syngeneic transplants (n ⫽ 6). Survival time was analyzed using the Kaplan–Meier curve. (A) Survival times of heart allografts. (B) Survival times of skin allografts.
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Osaka, Japan). The PCR amplification was performed using the following settings: 40 cycles of 95°C for 15 seconds, 56°C for 15 seconds, and 72°C for 45 seconds, followed by a final elongation step at 72°C for 10 minutes. The results were expressed using threshold cycle values (Ct) relative to the -actin gene. The primers used in this study were as follows: NF-B sense, CTGATGTGCA TCGGCAAGTGG; NF-b anti-sense, CAGAAGTTGAGTTTC GGGTAGGC; -actin sense, CCGTGAAAAGATGACCCAG; -actin anti-sense, TAGCCACGCTCGGTCAGG.
679 was detected by chemiluminescence. The images were taken using a film cassette and exposed to x-ray film for 2–5 minutes.
Data Analysis Graft survival was analyzed using the Kaplan–Meier log-rank test. Other data were analyzed using the Student t test. A statistically significant difference was considered to be P ⬍ .05.
Electrophoretic Mobility Shift Assay
RESULTS Administration of T gondii STAg Prolonged the Survival of Allogenic Heart and Skin Grafts in Mice
The nuclear extract from the heart allografts was prepared with the NE-PER nuclear extraction reagent (Pierce, Rockford, Ill). The nuclear extract was centrifuged for 5 minutes at 10,000 g and stored at ⫺80°C. The probe with the sequence 5=-AGTTGAGGGGACT TTCCCAGGC-3= and its complementary strand were used. A nonradioactive method in which the 3= end of the probe was labeled with biotin was used for these experiments (Pierce). Following separation on a 4% nondenaturing gel, the samples were transferred to a positively charged nylon membrane. The membrane was ultraviolet cross-linked, and the biotin end-labeled probe
C57BL/6 (H-2b) mice received complete major histocompatibility complex-mismatched grafts from BALB/c (H-2d) mice. The recipients were divided into a control group receiving phosphate-buffered saline (PBS), a treatment group receiving 5 g STAg and a syngeneic control group. As shown in Fig 1A, the survival time of the heart allografts in the STAg-treated group was significantly longer than that of the PBS-treated group (85.17 ⫾ 14.06 vs 6.33 ⫾ 0.52 days, respectively; P ⬍ .05). Figure 1B shows that the
Fig 2. Immunohistologic analysis of the cardiac and skin allografts in the recipient mice after STAg treatment. Heart and skin transplantations were performed 4 days after the recipient mice were treated with STAg, and the grafts were harvested 7 days after transplantation. (A) The analysis of the cardiac allograft by H & E staining of paraffin sections (original magnification, ⫻400) and immunohistochemical staining for CD4⫹ and CD8⫹ T cells (original magnification, ⫻400). (D) The immunohistochemical staining of the CD4⫹ and CD8⫹ T cells from the skin allografts (original magnification, ⫻400). (B, C, E, F) The quantification of the cardiac and skin intragraft CD4⫹ and CD8⫹ T cells by counting all positively staining cells in four different high-power fields (⌸:P ⬍ .05). Results are representative of 3 similar experiments.
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Fig 2. (continued)
survival time of the skin allografts in the STAg-treated group was also significantly longer than that of the PBStreated group (24.17 ⫾ 2.32 vs 7.83 ⫾ 0.75 days, respectively; P ⬍ .05). These data indicated that T gondii STAg strongly protected the mouse cardiac and skin allografts against rejection. T gondii STAg Reduced the Lymphocyte Infiltration in Cardiac and Skin Allografts
The grafts were harvested 7 days after heart transplantation or 8 days after skin transplantation. The histopathologic and immunohistochemical staining showed that the grafts from the control group had more lymphocyte infiltration and tissue damage than the grafts from the STAg-treated group. The immunohistological staining of CD4⫹ T cells and CD8⫹ T cells is shown in Fig 2. The mean number of T cells per high-power field represented the degree of rejection.
peripheral blood serum. We harvested the blood at different times posttransplant and measured the cytokine levels by Luminex. As shown in Fig 3, IL-12, IL-2, and IL-17 concentrations were much lower in the mice treated with STAg than those in the PBS-treated group, whereas IL-10 was higher in the STAg-treated mice than in the PBStreated group. The NF-B Pathway Was Activated in the Allografts After STAg Treatment
Because NF-B can activate the secretion of inflammatory cytokines, we speculated that the NF-B signal might be suppressed after STAg treatment. To evaluate this hypothesis, we analyzed the expression of NF-B in the allografts. Surprisingly, after STAg treatment, NF-B expression was higher 7 days posttransplant than in the PBS-treated group and persisted for a long period of time (Fig 4A–C). DISCUSSION
Decreased Serum Levels of the Proinflammatory Cytokines IL-12, IL-17, and IL-2, and Increased IL-10 Levels in the Allograft Recipients Treated With STAg
To explore the mechanism by which STAg protects against allograft rejection, we analyzed the level of cytokines in the
Graft rejection and the multiple side effects associated with immunosuppressive treatment are the most challenging problems that arise after transplantation. There is a strong need for novel drugs with few side effects or rejectionsuppressing therapies. Ledingham et al7 and Liwski et al8
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Fig 3. Changes in cytokine levels in the serum of STAg-treated mice posttransplantation. The recipient mice treated with STAg or PBS were humanely killed at different times posttransplant, and serum was collected from the peripheral blood. The cytokine levels were measured using a Luminex assay (PBS-treated group, n ⫽ 3; STAg-treated group; n ⫽ 4; ⌸, P ⬍ .05). (A) Heart and (B) skin transplants.
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Fig 4. NF-B activity in the allografts after STAg treatment. Heart grafts were harvested at different time points posttransplantation. Several methods were used to measure the expression of NF-B p65, all of which indicated that NF-B p65 was activated in the allografts treated with STAg. Results of the (A) Western blot, (B) EMSA, and (C) PCR analyses. Results are representative of 3 similar experiments.
discovered that infection by Nippostrongylus brasiliensis could prolong the survival time of kidney and heart allografts in mice, but the mechanism was not fully explored. In this study, we found that T gondii STAg can significantly prolong the survival time of heart and skin allografts to 85.17 ⫾ 14.06 and 24.17 ⫾ 2.32 days, respectively. T gondii is an intracellular protozoan parasite that can trigger a strong Th1 response during parasite replication.9 An uncontrolled Th1 response can cause severe symptoms in the host, such as acute inflammation, necrotic tissue damage, and eventual host death.10 Interestingly, most hosts infected by T gondii first experience an acute phase of infection and then enter a long-term chronic stage, in which their immune system tolerates the presence of the parasite. Butcher demonstrated that T gondii infection can induce rapid IB phosphorylation and degradation, but the activated NF-B subunits failed to translocate to the nucleus. This impairment inhibited the induction of proinflammatory cytokines, and the infected cells subsequently subjected
WANG, FANG, HUANG ET AL
to LPS treatment had severely impaired IL-12 production.11 In this study, we discovered that NF-B P65 was activated in the heart allografts of mice treated with STAg at a higher level than in the PBS-treated control allografts. The same trend was observed at both the protein and transcriptional levels. The heart allografts from the mice treated with STAg were only slightly rejected and could survive longer than those from the mice in the PBS-treated group. IL-10, a cytokine with broad immune regulatory functions, is expressed in Th2 cells and in a variety of other cells12,13 and is thought to play a protective role during infection with T gondii or other parasites that also induce a strong Th1 response.14 –16 Traditionally, the IL-10 that regulates T-cell effector function was thought to be of paracrine origin. During T gondii infection, mice with a targeted deletion of IL-10 production in T cells recapitulated the phenotype observed in mice constitutively lacking IL-10.17 In our study, we discovered that IL-10 is present in the serum of mice treated with STAg at a much higher level 3 and 7 days posttransplant than in mice treated with PBS, and these elevated levels last for about 4 weeks. IL-2, IL-12, and IL-17 levels were lower in the STAg-treated group compared with the PBS-treated group 7 days posttransplant. In conclusion, our results show that T gondii STAg significantly prolongs the survival time of heart and skin transplants. Compared with the control group, lymphocyte infiltration was strongly reduced in the STAg-treated animals and was accompanied by a reduction in the level of proinflammatory cytokines, indicating that this model of parasite infection may inhibit the adaptive immune response.
REFERENCES 1. Dantal J, Soulillou JP: Immunosuppressive drugs and the risk of cancer after organ transplantation. N Engl J Med 352:1371, 2005 2. Hunter CA, Bermudez L, Beernink H, et al: Transforming growth factor-beta inhibits interleukin-12-induced production of interferon-gamma by natural killer cells: a role for transforming growth factor-beta in the regulation of T cell-independent resistance to Toxoplasma gondii. Eur J Immunol 25:994, 1995 3. Neyer LE, Grunig G, Fort M, et al: Role of interleukin-10 in regulation of T-cell-dependent and T-cell-independent mechanisms of resistance to Toxoplasma gondii. Infect Immun 65:1675, 1997 4. Langermans JA, Nibbering PH, Van Vuren-Van Der Hulst ME, et al: Transforming growth factor-beta suppresses interferongamma-induced toxoplasmastatic activity in murine macrophages by inhibition of tumour necrosis factor-alpha production. Parasite Immunol 23:169, 2001 5. Zeng Z, Ming C, Zhen Z, et al: Can an allograft become harmonious with its recipient by analogous mechanisms to the long-term survival of a parasite in its host? Transplantation is similar to parasitism and chronic parasite infection can prolong allograft survival. Med Hypotheses 65:1047, 2005 6. Chen ZH: A technique of cervical heterotopic heart transplantation in mice. Transplantation 52:1099, 1991 7. Ledingham DL, McAlister VC, Ehigiator HN, et al: Prolongation of rat kidney allograft survival by nematodes. Transplantation 61:184, 1996
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8. Liwski R, Zhou J, McAlister V, et al: Prolongation of allograft survival by Nippostrongylus brasiliensis is associated with decreased allospecific cytotoxic T lymphocyte activity and development of T cytotoxic cell type 2 cells. Transplantation 69:1912, 2000 9. Gazzinelli RT, Wysocka M, Hayashi S, et al: Parasite-induced IL-12 stimulates early IFN-gamma synthesis and resistance during acute infection with Toxoplasma gondii. J Immunol 153:2533, 1994 10. Gazzinelli RT, Wysocka M, Hieny S, et al: In the absence of endogenous IL-10, mice acutely infected with Toxoplasma gondii succumb to a lethal immune response dependent on CD4⫹ T cells and accompanied by overproduction of IL-12, IFN-gamma and TNF-alpha. J Immunol 157:798, 1996 11. Oldenhove G, Bouladoux N, Wohlfert EA, et al: Decrease of Foxp3⫹ Treg cell number and acquisition of effector cell phenotype during lethal infection. Immunity 31:772, 2009 12. Fiorentino DF, Bond MW, Mosmann TR: Two types of mouse T helper cell. IV. Th2 clones secrete a factor that inhibits cytokine production by Th1 clones. J Exp Med 170:2081, 1989
683 13. Moore KW, O’Garra A, de Waal Malefyt R, et al: Interleukin10. Annu Rev Immunol 11:165, 1993 14. Suzuki Y, Sher A, Yap G, et al: IL-10 is required for prevention of necrosis in the small intestine and mortality in both genetically resistant BALB/c and susceptible C57BL/6 mice following peroral infection with Toxoplasma gondii. J Immunol 164:5375, 2000 15. Deckert M, Soltek S, Geginat G, et al: Endogenous interleukin-10 is required for prevention of a hyperinflammatory intracerebral immune response in Listeria monocytogenes meningoencephalitis. Infect Immun 69:4561, 2001 16. Oakley OR, Garvy BA, Humphreys S, et al: Increased weight loss with reduced viral replication in interleukin-10 knock-out mice infected with murine cytomegalovirus. Clin Exp Immunol 151:155, 2008 17. Roers A, Siewe L, Strittmatter E, et al: T cell-specific inactivation of the interleukin 10 gene in mice results in enhanced T cell responses but normal innate responses to lipopolysaccharide or skin irritation. J Exp Med 200:1289, 2004
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© 2013 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 45, 677– 683 (2013)
We previously observed that T gondii infection before transplantation significantly prolonged the survival time of allografts in rats while simultaneously suppressing inflammation. We hypothesize that transplantation is similar to parasitism and that chronic parasite infection could prolong allograft survival.5 From the Institute of Organ Transplantation (S.W., Ze.F., X.H., X.L., N.G., C.M.), Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Key Laboratory of Ministry of Health, Key Laboratory of Ministry of Education, Wuhan, China; and Department of Parasite (Z.F.), Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China. This work was supported by the National Natural Science and Foundation of China, grant 30772052. Address correspondence to Changsheng Ming, MD, PhD, Institute of Organ Transplantation, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China. E-mail: [email protected] 0041-1345/–see front matter http://dx.doi.org/10.1016/j.transproceed.2012.02.036 677
678
OBJECTIVE
In this study, we investigated whether soluble tachyzoite antigen (STAg) from T gondii has the same protective effects as the whole parasite on mouse heart and skin allografts and explored the mechanisms that are involved in this protection. MATERIALS AND METHODS Parasites and STAg The T gondii (RH strain) was a gift from the Parasite Division of Tongji Medical School and was used to infect BALB/c (H-2d) mice. STAg was isolated from T gondii (RH strain) tachyzoites grown in infected BALB/c (H-2d) mice. In brief, the peritoneal lavage fluid of the infected mice was purified by filtration through a 5-m diameter cellulose membrane. After sonication, the samples were centrifuged at 9000 g for 30 minutes. The resulting supernatant is referred to as T gondii STAg.
Animal Treatment and Cardiac or Skin Transplantation C57BL/6 (H-2b) mice were injected subcutaneously with 5 g of STAg 4 days before cardiac or skin transplantation. The cardiac transplantation was performed as previously reported.6 Briefly, after the recipient mice were anesthetized, the cardiac grafts obtained from BALB/c (H-2d) or C57BL/6 (H-2b) mice were transplanted into the cervical position of the recipient mice, with end-to-end anastomoses between the donor heart aorta and recipient carotid artery, and the donor pulmonary artery and recipient jugular vein. The total cessation of cardiac contraction was defined as the endpoint. Full tail skin was harvested from the sacrificed BALB/c (H-2d) mice. The skin was cut to a size of 8 ⫻ 8 mm. The C57BL/6 (H-2b) mice were anesthetized and shaved around the chest and backside, and a 9 ⫻ 9 mm square of skin was removed from the backside. The skin allografts were fixed on the graft beds with adhesive plaster. The rejection was diagnosed by cyanosis, erythema, erosion, and loss of the skin graft.
WANG, FANG, HUANG ET AL incubated for 2 hours, after which the biotinylated detector antibody was added. The plates were then washed and streptavidinRPE was added. After a final wash, the plates were analyzed using a Luminex 200 instrument.
Western Blot Analysis Equal amounts of tissue protein (30 g) per lane were electrophoresed on a 12% SDS-PAGE gel for separation and then transferred to PVDF membranes. After blocking, the membranes were incubated with primary antibodies against nuclear factor (NF)-B or -actin overnight at 4°C. The membranes were then incubated with a secondary antibody (HRP-conjugated goat anti-rabbit IgG). The peroxidase activity was detected using the ECL assay and analyzed by densitometry and image software.
Quantitative Real-Time Polymerase Chain Reaction Total RNA was extracted from the heart grafts using Trizol (Invitrogen, Grand Island, NY) as per the manufacturer’s protocol. Quantitative real-time polymerase chain reactions (qRT-PCRs) were performed using Rotor Gene 3000 (Corbett Research, Sydney, Australia) with a Quantitect SYBR Green PCR Kit (Toyobo,
Histopathology and Immunohistochemistry Allograft samples for histology were processed routinely by the histology laboratory. The tissue was fixed in 10% formalin and then embedded in paraffin before the preparation of sections. The tissue sections were stained with hematoxylin and eosin (H & E). Histologic rejection was evaluated by ⱖ2 experienced pathologists blinded to the source of the experimental animals and histology tissues. Immunohistochemical staining was performed on 4-mthick sections cut from formalin-fixed, paraffin-embedded tissue. Briefly, after deparaffinization and hydration, the sections were blocked with 3% H2O2 and 10% normal goat serum. The sections were incubated with a primary antibody overnight at 4°C. After washing, the sections were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin (Ig)G for 30 minutes at 37°C, followed by visualization using DAB.
Luminex Analysis of the Cytokines in the Serum For the Luminex assay, 25 L of the diluted bead solution was added to each well, after which the beads were washed. Next, 50 L of the incubation buffer and 100 L of the appropriate standard dilution was added to the wells designated for the standard curve, and 50 L of the assay diluent followed by 50 L of the sample was added to the wells designated for the samples. The samples were
Fig 1. Survival times of the heart and skin allografts after STAg treatment. The heterotopic heart transplants from the BALB/c (H-2d) donor mice to the C57BL/6 (H-2b) recipients were monitored daily by manual palpation. The rejection of the cardiac grafts was defined as the cessation of graft pulsing. The rejection of the skin transplants from the BALB/c (H-2d) donor mice to the C57BL/6 (H-2b) recipients was defined as the presence of cyanosis, erythema, erosion, and loss of the skin graft. The recipient mice were divided into groups receiving PBS (control group, n ⫽ 6), 5 g STAg (n ⫽ 6), or syngeneic transplants (n ⫽ 6). Survival time was analyzed using the Kaplan–Meier curve. (A) Survival times of heart allografts. (B) Survival times of skin allografts.
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Osaka, Japan). The PCR amplification was performed using the following settings: 40 cycles of 95°C for 15 seconds, 56°C for 15 seconds, and 72°C for 45 seconds, followed by a final elongation step at 72°C for 10 minutes. The results were expressed using threshold cycle values (Ct) relative to the -actin gene. The primers used in this study were as follows: NF-B sense, CTGATGTGCA TCGGCAAGTGG; NF-b anti-sense, CAGAAGTTGAGTTTC GGGTAGGC; -actin sense, CCGTGAAAAGATGACCCAG; -actin anti-sense, TAGCCACGCTCGGTCAGG.
679 was detected by chemiluminescence. The images were taken using a film cassette and exposed to x-ray film for 2–5 minutes.
Data Analysis Graft survival was analyzed using the Kaplan–Meier log-rank test. Other data were analyzed using the Student t test. A statistically significant difference was considered to be P ⬍ .05.
Electrophoretic Mobility Shift Assay
RESULTS Administration of T gondii STAg Prolonged the Survival of Allogenic Heart and Skin Grafts in Mice
The nuclear extract from the heart allografts was prepared with the NE-PER nuclear extraction reagent (Pierce, Rockford, Ill). The nuclear extract was centrifuged for 5 minutes at 10,000 g and stored at ⫺80°C. The probe with the sequence 5=-AGTTGAGGGGACT TTCCCAGGC-3= and its complementary strand were used. A nonradioactive method in which the 3= end of the probe was labeled with biotin was used for these experiments (Pierce). Following separation on a 4% nondenaturing gel, the samples were transferred to a positively charged nylon membrane. The membrane was ultraviolet cross-linked, and the biotin end-labeled probe
C57BL/6 (H-2b) mice received complete major histocompatibility complex-mismatched grafts from BALB/c (H-2d) mice. The recipients were divided into a control group receiving phosphate-buffered saline (PBS), a treatment group receiving 5 g STAg and a syngeneic control group. As shown in Fig 1A, the survival time of the heart allografts in the STAg-treated group was significantly longer than that of the PBS-treated group (85.17 ⫾ 14.06 vs 6.33 ⫾ 0.52 days, respectively; P ⬍ .05). Figure 1B shows that the
Fig 2. Immunohistologic analysis of the cardiac and skin allografts in the recipient mice after STAg treatment. Heart and skin transplantations were performed 4 days after the recipient mice were treated with STAg, and the grafts were harvested 7 days after transplantation. (A) The analysis of the cardiac allograft by H & E staining of paraffin sections (original magnification, ⫻400) and immunohistochemical staining for CD4⫹ and CD8⫹ T cells (original magnification, ⫻400). (D) The immunohistochemical staining of the CD4⫹ and CD8⫹ T cells from the skin allografts (original magnification, ⫻400). (B, C, E, F) The quantification of the cardiac and skin intragraft CD4⫹ and CD8⫹ T cells by counting all positively staining cells in four different high-power fields (⌸:P ⬍ .05). Results are representative of 3 similar experiments.
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Fig 2. (continued)
survival time of the skin allografts in the STAg-treated group was also significantly longer than that of the PBStreated group (24.17 ⫾ 2.32 vs 7.83 ⫾ 0.75 days, respectively; P ⬍ .05). These data indicated that T gondii STAg strongly protected the mouse cardiac and skin allografts against rejection. T gondii STAg Reduced the Lymphocyte Infiltration in Cardiac and Skin Allografts
The grafts were harvested 7 days after heart transplantation or 8 days after skin transplantation. The histopathologic and immunohistochemical staining showed that the grafts from the control group had more lymphocyte infiltration and tissue damage than the grafts from the STAg-treated group. The immunohistological staining of CD4⫹ T cells and CD8⫹ T cells is shown in Fig 2. The mean number of T cells per high-power field represented the degree of rejection.
peripheral blood serum. We harvested the blood at different times posttransplant and measured the cytokine levels by Luminex. As shown in Fig 3, IL-12, IL-2, and IL-17 concentrations were much lower in the mice treated with STAg than those in the PBS-treated group, whereas IL-10 was higher in the STAg-treated mice than in the PBStreated group. The NF-B Pathway Was Activated in the Allografts After STAg Treatment
Because NF-B can activate the secretion of inflammatory cytokines, we speculated that the NF-B signal might be suppressed after STAg treatment. To evaluate this hypothesis, we analyzed the expression of NF-B in the allografts. Surprisingly, after STAg treatment, NF-B expression was higher 7 days posttransplant than in the PBS-treated group and persisted for a long period of time (Fig 4A–C). DISCUSSION
Decreased Serum Levels of the Proinflammatory Cytokines IL-12, IL-17, and IL-2, and Increased IL-10 Levels in the Allograft Recipients Treated With STAg
To explore the mechanism by which STAg protects against allograft rejection, we analyzed the level of cytokines in the
Graft rejection and the multiple side effects associated with immunosuppressive treatment are the most challenging problems that arise after transplantation. There is a strong need for novel drugs with few side effects or rejectionsuppressing therapies. Ledingham et al7 and Liwski et al8
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Fig 3. Changes in cytokine levels in the serum of STAg-treated mice posttransplantation. The recipient mice treated with STAg or PBS were humanely killed at different times posttransplant, and serum was collected from the peripheral blood. The cytokine levels were measured using a Luminex assay (PBS-treated group, n ⫽ 3; STAg-treated group; n ⫽ 4; ⌸, P ⬍ .05). (A) Heart and (B) skin transplants.
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Fig 4. NF-B activity in the allografts after STAg treatment. Heart grafts were harvested at different time points posttransplantation. Several methods were used to measure the expression of NF-B p65, all of which indicated that NF-B p65 was activated in the allografts treated with STAg. Results of the (A) Western blot, (B) EMSA, and (C) PCR analyses. Results are representative of 3 similar experiments.
discovered that infection by Nippostrongylus brasiliensis could prolong the survival time of kidney and heart allografts in mice, but the mechanism was not fully explored. In this study, we found that T gondii STAg can significantly prolong the survival time of heart and skin allografts to 85.17 ⫾ 14.06 and 24.17 ⫾ 2.32 days, respectively. T gondii is an intracellular protozoan parasite that can trigger a strong Th1 response during parasite replication.9 An uncontrolled Th1 response can cause severe symptoms in the host, such as acute inflammation, necrotic tissue damage, and eventual host death.10 Interestingly, most hosts infected by T gondii first experience an acute phase of infection and then enter a long-term chronic stage, in which their immune system tolerates the presence of the parasite. Butcher demonstrated that T gondii infection can induce rapid IB phosphorylation and degradation, but the activated NF-B subunits failed to translocate to the nucleus. This impairment inhibited the induction of proinflammatory cytokines, and the infected cells subsequently subjected
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to LPS treatment had severely impaired IL-12 production.11 In this study, we discovered that NF-B P65 was activated in the heart allografts of mice treated with STAg at a higher level than in the PBS-treated control allografts. The same trend was observed at both the protein and transcriptional levels. The heart allografts from the mice treated with STAg were only slightly rejected and could survive longer than those from the mice in the PBS-treated group. IL-10, a cytokine with broad immune regulatory functions, is expressed in Th2 cells and in a variety of other cells12,13 and is thought to play a protective role during infection with T gondii or other parasites that also induce a strong Th1 response.14 –16 Traditionally, the IL-10 that regulates T-cell effector function was thought to be of paracrine origin. During T gondii infection, mice with a targeted deletion of IL-10 production in T cells recapitulated the phenotype observed in mice constitutively lacking IL-10.17 In our study, we discovered that IL-10 is present in the serum of mice treated with STAg at a much higher level 3 and 7 days posttransplant than in mice treated with PBS, and these elevated levels last for about 4 weeks. IL-2, IL-12, and IL-17 levels were lower in the STAg-treated group compared with the PBS-treated group 7 days posttransplant. In conclusion, our results show that T gondii STAg significantly prolongs the survival time of heart and skin transplants. Compared with the control group, lymphocyte infiltration was strongly reduced in the STAg-treated animals and was accompanied by a reduction in the level of proinflammatory cytokines, indicating that this model of parasite infection may inhibit the adaptive immune response.
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