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Bacteriophages and Transplantation Tolerance
Bacteriophages and Transplantation Tolerance A. Górski, M. Kniotek, A. Perkowska-Ptasin´ska, A. Mróz, A. Przerwa, W. Gorczyca, K. Da˛browska, B. Weber-Da˛browska, and M. Nowaczyk ABSTRACT Our recent findings suggest that bacteriophages (phages) may not only eliminate bacteria, but also modulate immune functions. In this communication, we demonstrate that phages may strongly inhibit human T-cell activation and proliferation as well as activation of the nuclear transcription factor NF-kappaB in response to a viral pathogen. Phage administration in vivo can diminish cellular infiltration of allogeneic skin allografts. Thus, phage treatment should be considered in antibiotic-resistant posttransplantation infections. Furthermore, phages could find a broader application in clinical transplantation.
O
UR RECENT FINDINGS strongly suggest that bacteriophages (phages), viral predators that efficiently eliminate bacteria, may have a much broader immunobiological role and contribute to the body’s defenses against internal and external assault.1 Some phages can exert immunosuppressive action; in fact, our preliminary data indicate that phages may extend skin allograft survival in mice.2 Here we provide more data suggesting that phages could contribute to transplantation tolerance. MATERIALS AND METHODS Balb/c mice received skin transplants from C57B1 donors. On each consecutive day posttransplantation, the recipients were injected intraperitoneally with 0.1 mL of PBS (control) or purified T4 phages (2 ⫻ 105 PFU/mice). The transplants were removed and processed for standard histological examination (fixed in formalin, embedded in paraffin, and stained with hematoxylin-eosin). Two experienced pathologists independently examined the intensity of graft infiltration using a scale from 0 (no infiltration) to ⫹3 (very intense infiltration). Phage effects on in vitro T-cell activation triggered via CD3 receptor complex were evaluated using a standard cell culture system with OKT3 monoclonal antibody (Ortho) immobilized on plastic plates and thymidine incorporation.3 Phages were coimmobilized on plates or added to lymphocyte cultures in suspension. The effect of phages on the nuclear transcription factor (NFkappaB) activity was evaluated in human umbilical vein endothelial cells (HUVEC) and human epithelial cell line A549. Phages T4 as well as HSV-1 virus were used as NF-kappaB-inducing agents. The HUVEC and human epithelial-like lung cancer line A549 (ATCC CCL 185) cells were incubated for 8 hours at 37°C either in the absence (control) or in the presence of phage T4 (102–103 PFU/ cell). Virus HSV-1 (2 virions/cell) was added after 1 hour of preincubation with or without the phage, and the cells were incubated for 7 consecutive hours. Then the NF-B activity was © 2006 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 38, 331–333 (2006)
determined by means of electromobility shift assay (EMSA),4 quantified densitometrically and expressed as percent (mean ⫾ SD) of the activity measured in untreated (control) cells. The Student t test was used for evaluation of differences in the NF-B activity between cells treated with HSV-1 and cells treated either with HSV-1 in the presence of phage T4 or with phage T4 only.
RESULTS AND DISCUSSION
Microscopic evaluation of the skin transplants removed on days 1 to 3 posttransplantation revealed only scattered, focally polymorphic (mononuclear cells and neutrophils) dermal infiltration in both the control and phage-treated group. On later days, the differences in graft infiltration between the controls and the experimental group increased and were most striking on days 7 and 8 posttransplantation in that phages tended to reduce the homing of mostly mononuclear cells, and, to some extent, also neutrophils to the graft site (Fig 1). Figure 2 shows that T4 phages strongly inhibit human T-cell activation; the effect is stronger when phages are present in the culture medium, although phages coated on From the Transplantation Institute, Warsaw Medical University, Warsaw, Poland (A.G., M.K., A.P.-P., A.M., A.P., M.N.) and the L. Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland (A.G., W.G., K.D., B.W.-D.). This work was supported by The State Committee for Research grants PBZ-MIN-007/P04/2003, 2P05B 04727, and Warsaw Medical University intramural grant 1MG/W1. Address reprint requests to M. Nowaczyk, Transplantation Institute, Warsaw Medical University, 02-006 Warsaw, Poland. E-mail: [email protected] 0041-1345/06/$–see front matter doi:10.1016/j.transproceed.2005.12.073 331
GÓRSKI, KNIOTEK, PERKOWSKA-PTASIN´SKA ET AL
332 Table 1. Effect of HSV-1 Virus and T4 Phage on NF-B Activity NF-B Activity Treatment
HUVEC
A549
Untreated (control) HSV-1 HSV-1 ⫹ T4
100 293.7 ⫾ 23.7 101.0 ⫾ 27.2 (P ⬍ .0002) 114.2 ⫾ 21.0
100 295.0 ⫾ 4.2 134.7 ⫾ 25.8 (P ⬍ .001) 112.8 ⫾ 34.5
T4
Data presented as percent (mean ⫾ SD) of the activity in untreated (control) cells. P values for differences between cells treated with HSV-1 and cells treated with HSV-1 in the presence of T4 phage are shown.
plates containing immobilized OKT activator are also inhibitory. In contrast to HSV-1 virus, phages do not cause significant activation of NF-kappaB. Moreover, when the cells are preincubated with phages the ability of the pathogenic virus to induce NF-kappaB activity was abolished (Table 1). These results confirm and extend our earlier findings suggesting that phages could be beneficial in clinical trans-
Fig 1. Skin allografts from mice receiving bacteriophages (A) and Saline (B). H/E stain, 10⫻ magnification. Day ⫹8 posttransplant.
Fig 2. Bacteriophage-mediated inhibition of T cell proliferation triggered via CD3 complex: (A) Phage concentration 1010/mL r ⫽ (B) 10a/mL (lymphocyte/phage ratio 1:1000 and 1:100, respectively). White columns indicate phages added to the culture medium in suspension; Black columns indicate phages coated onto plates with immobilized OKT3. Bars show SD.
plantation. They could help in treating bacterial infections (and perhaps also viral infections, as suggested by us recently,5 reduce the inflammatory infiltration of the graft that may lead to its injury and eventual loss, and directly inhibit allograft-induced T-cell activation. Phage-mediated inhibition of NF-kappaB activation seems to be another important factor contributing to the tolerating action of phages in transplantation. This factor is a key regulator of transcription after TCR and costimulatory receptor ligation; mice expressing an inhibitor of NF-kappaB permanently accept fully allogeneic skin and secondary donor skin grafts. Thus, transplantation tolerance may be dependent on defective T-cell-intrinsic NF-kappaB activation.6 Inhibition of NF-kappaB activation ameliorates lung injury during acute allograft rejection in rats and reduces inflammation processes in lung transplantation,7 an effect similar to our histologic findings in skin grafts of mice treated with T4 phages. The head of these phages contains a highly immunogenic outer capsid protein (Hoc), its most prominent surface feature, being an extended molecule with a rounded base from which a thin neck and a globular head protrude.8 Interestingly, Hoc protein contains an Ig superfamily domain.9 The T4 immunomodulatory effects described in the present paper as well as our earlier reports10 may at least partly depend on Hoc, since the effects of the mutant HAP1 with defective Hoc expression are much weaker (Przerwa et al, unpublished observations). Initial results with phage treatment of urinary tract infections in renal allograft recipients are encouraging.11 The present report strongly
BACTERIOPHAGES AND TRANSPLANTATION TOLERANCE
suggests that phages should find a much broader application in clinical transplantation. REFERENCES 1. Górski A, Weber-Da˛browska B: The potential role of endogenous bacteriophages in controlling invading pathogens. Cell Mol Life Sci 62:511, 2005 2. Górski A, Nowaczyk M, Weber-Da˛browska B, et al: New insights into the possible role of bacteriophages in transplantation. Transplant Proc 35:2372, 2003 3. Górski A, Grieb P, Makula J, et al: 2-Chloro-2-deoxyadenosine— a novel immunosuppressive agent. Transplantation 56:1253, 1993 4. Guo M, Sahni SK, Francis CW: Fibrinogen regulates the expression of inflammatory chemokines through NF-kappaB activation of endothelial cells. Thromb Haemost 92:858, 2004 5. Mie˛dzybrodzki R, Fortuna W, Weber-Da˛browska B, et al: Bacterial viruses against viruses pathogenic for man? Virus Res 110:1, 2005
333 6. Zhou P, Balin SJ, Mashayekhi M, et al: Transplantation tolerance in NF-kappaB-impaired mice is not due to regulation but is prevented by transgenic expression of Bcl-xL. J Immunol 174: 3447, 2005 7. Ohmori K, Takeda S, Miyoshi S, et al: Attenuation of lung injury in allograft rejection using NF-kappaB decoy transfection— novel strategy for use in lung transplantation. Eur J Cardiothorac Surg 27:23, 2005 8. Olson NH, Gingery F, Eiserling FA, et al: The structure of isometric capsids of bacteriophage T4. Virology 279:385, 2001 9. Bateman A, Eddy S, Mesyanzhinov VV: A member of the immunoglobulin superfamily in bacteriophage T4. Virus Genes 14:163, 1997 10. Kniotek M, Weber-Da˛browska B, Da˛browska K, et al: Phages as immunomodulators of antibody production. In: Genomic Issues, Immune System Activation and Allergy. Immunology 2004. Bologna, Italy: Monduzzi Editors; 2004, p 33 11. Boratyn ´ska M, Szewczyk Z, Weber-Da˛browska B: The clinical evaluation of bacteriophage treatment of urinary tract infections [in Polish]. Adv Clin Exp Med 3:7, 1994
O
UR RECENT FINDINGS strongly suggest that bacteriophages (phages), viral predators that efficiently eliminate bacteria, may have a much broader immunobiological role and contribute to the body’s defenses against internal and external assault.1 Some phages can exert immunosuppressive action; in fact, our preliminary data indicate that phages may extend skin allograft survival in mice.2 Here we provide more data suggesting that phages could contribute to transplantation tolerance. MATERIALS AND METHODS Balb/c mice received skin transplants from C57B1 donors. On each consecutive day posttransplantation, the recipients were injected intraperitoneally with 0.1 mL of PBS (control) or purified T4 phages (2 ⫻ 105 PFU/mice). The transplants were removed and processed for standard histological examination (fixed in formalin, embedded in paraffin, and stained with hematoxylin-eosin). Two experienced pathologists independently examined the intensity of graft infiltration using a scale from 0 (no infiltration) to ⫹3 (very intense infiltration). Phage effects on in vitro T-cell activation triggered via CD3 receptor complex were evaluated using a standard cell culture system with OKT3 monoclonal antibody (Ortho) immobilized on plastic plates and thymidine incorporation.3 Phages were coimmobilized on plates or added to lymphocyte cultures in suspension. The effect of phages on the nuclear transcription factor (NFkappaB) activity was evaluated in human umbilical vein endothelial cells (HUVEC) and human epithelial cell line A549. Phages T4 as well as HSV-1 virus were used as NF-kappaB-inducing agents. The HUVEC and human epithelial-like lung cancer line A549 (ATCC CCL 185) cells were incubated for 8 hours at 37°C either in the absence (control) or in the presence of phage T4 (102–103 PFU/ cell). Virus HSV-1 (2 virions/cell) was added after 1 hour of preincubation with or without the phage, and the cells were incubated for 7 consecutive hours. Then the NF-B activity was © 2006 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 38, 331–333 (2006)
determined by means of electromobility shift assay (EMSA),4 quantified densitometrically and expressed as percent (mean ⫾ SD) of the activity measured in untreated (control) cells. The Student t test was used for evaluation of differences in the NF-B activity between cells treated with HSV-1 and cells treated either with HSV-1 in the presence of phage T4 or with phage T4 only.
RESULTS AND DISCUSSION
Microscopic evaluation of the skin transplants removed on days 1 to 3 posttransplantation revealed only scattered, focally polymorphic (mononuclear cells and neutrophils) dermal infiltration in both the control and phage-treated group. On later days, the differences in graft infiltration between the controls and the experimental group increased and were most striking on days 7 and 8 posttransplantation in that phages tended to reduce the homing of mostly mononuclear cells, and, to some extent, also neutrophils to the graft site (Fig 1). Figure 2 shows that T4 phages strongly inhibit human T-cell activation; the effect is stronger when phages are present in the culture medium, although phages coated on From the Transplantation Institute, Warsaw Medical University, Warsaw, Poland (A.G., M.K., A.P.-P., A.M., A.P., M.N.) and the L. Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland (A.G., W.G., K.D., B.W.-D.). This work was supported by The State Committee for Research grants PBZ-MIN-007/P04/2003, 2P05B 04727, and Warsaw Medical University intramural grant 1MG/W1. Address reprint requests to M. Nowaczyk, Transplantation Institute, Warsaw Medical University, 02-006 Warsaw, Poland. E-mail: [email protected] 0041-1345/06/$–see front matter doi:10.1016/j.transproceed.2005.12.073 331
GÓRSKI, KNIOTEK, PERKOWSKA-PTASIN´SKA ET AL
332 Table 1. Effect of HSV-1 Virus and T4 Phage on NF-B Activity NF-B Activity Treatment
HUVEC
A549
Untreated (control) HSV-1 HSV-1 ⫹ T4
100 293.7 ⫾ 23.7 101.0 ⫾ 27.2 (P ⬍ .0002) 114.2 ⫾ 21.0
100 295.0 ⫾ 4.2 134.7 ⫾ 25.8 (P ⬍ .001) 112.8 ⫾ 34.5
T4
Data presented as percent (mean ⫾ SD) of the activity in untreated (control) cells. P values for differences between cells treated with HSV-1 and cells treated with HSV-1 in the presence of T4 phage are shown.
plates containing immobilized OKT activator are also inhibitory. In contrast to HSV-1 virus, phages do not cause significant activation of NF-kappaB. Moreover, when the cells are preincubated with phages the ability of the pathogenic virus to induce NF-kappaB activity was abolished (Table 1). These results confirm and extend our earlier findings suggesting that phages could be beneficial in clinical trans-
Fig 1. Skin allografts from mice receiving bacteriophages (A) and Saline (B). H/E stain, 10⫻ magnification. Day ⫹8 posttransplant.
Fig 2. Bacteriophage-mediated inhibition of T cell proliferation triggered via CD3 complex: (A) Phage concentration 1010/mL r ⫽ (B) 10a/mL (lymphocyte/phage ratio 1:1000 and 1:100, respectively). White columns indicate phages added to the culture medium in suspension; Black columns indicate phages coated onto plates with immobilized OKT3. Bars show SD.
plantation. They could help in treating bacterial infections (and perhaps also viral infections, as suggested by us recently,5 reduce the inflammatory infiltration of the graft that may lead to its injury and eventual loss, and directly inhibit allograft-induced T-cell activation. Phage-mediated inhibition of NF-kappaB activation seems to be another important factor contributing to the tolerating action of phages in transplantation. This factor is a key regulator of transcription after TCR and costimulatory receptor ligation; mice expressing an inhibitor of NF-kappaB permanently accept fully allogeneic skin and secondary donor skin grafts. Thus, transplantation tolerance may be dependent on defective T-cell-intrinsic NF-kappaB activation.6 Inhibition of NF-kappaB activation ameliorates lung injury during acute allograft rejection in rats and reduces inflammation processes in lung transplantation,7 an effect similar to our histologic findings in skin grafts of mice treated with T4 phages. The head of these phages contains a highly immunogenic outer capsid protein (Hoc), its most prominent surface feature, being an extended molecule with a rounded base from which a thin neck and a globular head protrude.8 Interestingly, Hoc protein contains an Ig superfamily domain.9 The T4 immunomodulatory effects described in the present paper as well as our earlier reports10 may at least partly depend on Hoc, since the effects of the mutant HAP1 with defective Hoc expression are much weaker (Przerwa et al, unpublished observations). Initial results with phage treatment of urinary tract infections in renal allograft recipients are encouraging.11 The present report strongly
BACTERIOPHAGES AND TRANSPLANTATION TOLERANCE
suggests that phages should find a much broader application in clinical transplantation. REFERENCES 1. Górski A, Weber-Da˛browska B: The potential role of endogenous bacteriophages in controlling invading pathogens. Cell Mol Life Sci 62:511, 2005 2. Górski A, Nowaczyk M, Weber-Da˛browska B, et al: New insights into the possible role of bacteriophages in transplantation. Transplant Proc 35:2372, 2003 3. Górski A, Grieb P, Makula J, et al: 2-Chloro-2-deoxyadenosine— a novel immunosuppressive agent. Transplantation 56:1253, 1993 4. Guo M, Sahni SK, Francis CW: Fibrinogen regulates the expression of inflammatory chemokines through NF-kappaB activation of endothelial cells. Thromb Haemost 92:858, 2004 5. Mie˛dzybrodzki R, Fortuna W, Weber-Da˛browska B, et al: Bacterial viruses against viruses pathogenic for man? Virus Res 110:1, 2005
333 6. Zhou P, Balin SJ, Mashayekhi M, et al: Transplantation tolerance in NF-kappaB-impaired mice is not due to regulation but is prevented by transgenic expression of Bcl-xL. J Immunol 174: 3447, 2005 7. Ohmori K, Takeda S, Miyoshi S, et al: Attenuation of lung injury in allograft rejection using NF-kappaB decoy transfection— novel strategy for use in lung transplantation. Eur J Cardiothorac Surg 27:23, 2005 8. Olson NH, Gingery F, Eiserling FA, et al: The structure of isometric capsids of bacteriophage T4. Virology 279:385, 2001 9. Bateman A, Eddy S, Mesyanzhinov VV: A member of the immunoglobulin superfamily in bacteriophage T4. Virus Genes 14:163, 1997 10. Kniotek M, Weber-Da˛browska B, Da˛browska K, et al: Phages as immunomodulators of antibody production. In: Genomic Issues, Immune System Activation and Allergy. Immunology 2004. Bologna, Italy: Monduzzi Editors; 2004, p 33 11. Boratyn ´ska M, Szewczyk Z, Weber-Da˛browska B: The clinical evaluation of bacteriophage treatment of urinary tract infections [in Polish]. Adv Clin Exp Med 3:7, 1994