- Email: [email protected]
Inhibition of Inflammatory Cytokine-Induced Response in Human Islet Cells by Withaferin A
Inhibition of Inflammatory Cytokine-Induced Response in Human Islet Cells by Withaferin A H. Peng, G. Olsen, Y. Tamura, H. Noguchi, S. Matsumoto, M.F. Levy, and B. Naziruddin
ABSTRACT Background. After islet cell transplantation, a substantial mass of islets are lost owing to nonspecific inflammatory reactions. Cytokine exposure before or after transplantation can upregulate expression of proinflammatory genes via the nuclear factor-B signaling pathway, eventually resulting in islet loss. Objective. To test the effects of a naturally occurring nuclear factor-B inhibitor, withaferin A, on regulation of inflammatory genes in human islets. Methods. Human pancreatic islets were isolated using a modified Ricordi protocol. Purified islets were cultured for 2 days. The effect of withaferin A treatment on islet cell viability was examined using the fluorescein diacetate-propidium iodide dye exclusion test, and on function using a static glucose stimulation assay. Islet cells were treated with a cytokine mixture (50 U/mL of interleukin-1, 1000 U/mL of tumor necrosis factor-␣, and 1000 U/mL of interferon-␥) for 48 hours with or without withaferin A, 1 g/mL. Treated islets were used for real-time polymerase chain reaction (PCR) array analysis for expression of inflammatory genes, and expression of other selected genes was analyzed using real-time PCR with single primers. Results. Glucose stimulation and viability assays demonstrated that withaferin A was not toxic to islet cells. Of 84 inflammation-related genes examined using real-time PCR array analysis, 9 were significantly upregulated by cytokine treatment compared with the control group. However, addition of withaferin A to the culture significantly inhibited expression of all genes. Conclusion. Withaferin A significantly inhibits the inflammatory response of islet cells with cytokine exposure. ype 1 diabetes mellitus is an autoimmune disease that leads to self-destruction of pancreatic beta cells that produce insulin. Islet cell transplantation to treat brittle forms of diabetes has achieved great success in recent years. However, immediately after transplantation, a substantial amount of islets are lost as a result of the instant bloodmediated inflammatory reaction, an innate immune response characterized by blood coagulation, platelet activation, complement reaction, and cytokine storm.1 In addition, the adaptive immune system slowly destroys the islet graft via lymphocyte infiltration and release of soluble mediators. In both processes, inflammatory cytokines have a crucial role by inducing apoptotic genes and chemokine gene expression in islet cells.2,3 Antibodies and receptor blockers have been used to reduce the effects of inflammatory cyto-
T
kines such as tumor necrosis factor-␣4; however, this strategy cannot entirely block islet graft loss in the long term. Previous studies have demonstrated that nuclear factor-B is the primary transcription factor responsible for expresFrom the Institute of Biomedical Studies, Baylor University, Waco (H.P., B.N.), the Baylor Regional Transplant Institute, Dallas and Fort Worth (G.O., Y.T., M.F.L., B.N.), and Baylor Research Institute, Islet Cell Laboratory, Fort Worth (H.N., S.M.), Texas. This study was supported in part by the Baylor Health Care System Foundation. Address reprint requests to Bashoo Naziruddin, PhD, Islet Cell Laboratory, Transplant Services Department, Baylor University Medical Center, 3500 Gaston Ave, Dallas, TX 75246. E-mail: [email protected]
0041-1345/10/$–see front matter doi:10.1016/j.transproceed.2010.05.131
© 2010 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
2058
Transplantation Proceedings, 42, 2058 –2061 (2010)
WITHAFERIN A INHIBITS ISLET CELL INFLAMMATORY RESPONSE
2059
sion of many inflammatory genes after cytokine stimulation. Withaferin A is a steroidal lactone (Fig 1) derived from Withania somnifera, a plant that has been used for centuries to treat several inflammatory disorders.5 The objective of the present study was to test this naturally occurring antiinflammatory agent for its protective effect on islet cells after cytokine exposure.
transcription–PCR reaction was run using the MX3000 system (Stratagene Corp) with the following program: 95°C for 10 minutes, and 40 cycles of 95°C for 15 seconds and 60°C for 60 seconds. Cycle threshold data were uploaded to a Web-based PCR array data analysis (http://www.sabiosciences.com/pcr/arrayanalysis.php) to analyze the fold change compared with controls:
MATERIALS AND METHODS Islet Isolation
where Ct is cycle threshold, GOI is gene of interest, and HKG is house keeping gene. The procedure for single-primer gene assays was the same except that primers of particular genes other than the entire array plate were used.
Human pancreases designated for research were obtained from normoglycemic cadavers using the 2-layer method. Islets were isolated using a modification of the Ricordi method,6 and stained with dithizone to determine counts and purity. Viability was assessed using the fluorescein diacetate-propidium iodide dye exclusion test. Islets used for the experiments were 89% to 90% viable, with purity of 60% to 85%.
Cytokine and Withaferin A Treatment After isolation, islet cells were treated with a cytokine mixture (50 U/mL of interleukin-1, 1000 U/mL of tumor necrosis factor-␣, and 1000 U/mL of interferon-␥; R&D Systems, Inc, Minneapolis, Minnesota) or cytokine mixture plus withaferin A, 1 g/mL, for 48 hours at 37°C in 5% carbon dioxide (CO2). Nontreated islets were used as controls. All islet cells were cultured in CMRL-based culture medium.
Reverse Transcription–PCR Inflammation Array and Primer Assays After 48 hours islets were harvested and lysed with buffer (Stratagene Corp, La Jolla, California). RNA was purified using an Absolutely RNA Miniprep Kit (Stratagene Corp), and was reverse transcribed into complementary DNA using an RT2 FirstStrand Kit (SABiosciences Corp, Frederick, Maryland). Template cDNA was mixed with RT2 SYBR green/ROX PCR master mix (SABiosciences Corp), and added to each well of a human inflammation gene array plate (SABiosciences Corp). The reverse
Fig 1.
Structure of withaferin A.
Fold change ⫽ 2(⌬Ct [control] ⫺ ⌬Ct [experiment]), ⌬Ct ⫽ Ct (GOI) ⫺ Ct (HKG)
Static Incubation Islet potency was tested using a static glucose-stimulated insulinrelease assay. Islets, 200 IEq, were cultured overnight at 37°C in 5% CO2. Islets in quadruplicates of 50 IEq were stimulated in low-glucose culture medium (2.8 mmol/L) at 37°C in 5% CO2 for 60 minutes. Later, the same islets were stimulated in high-glucose culture medium (20 mmol/L) under the same conditions. Insulin released in both cultures was measured using a human insulin enzyme-linked immunosorbent assay kit (Alpco Diagnostics, Salem, New Hampshire).
RESULTS
Islet cells treated with cytokine or cytokine plus withaferin A, and untreated islet cells were tested for inflammatory gene expression using a real-time PCR array. Of the 84 examined genes, 9 were markedly upregulated compared with untreated islets (Fig 2A): These genes were RANTES (CCL5; 2174-fold), IP10 (CXCL10; 24,255-fold), MIG (CXCL9; 8169-fold), ITAC (CXCL11; 3864-fold), TNF␣(43fold), IL1 (268-fold), MCP-2 (CCL8; 4891-fold), CXCL5 (599-fold), and IL8 (179-fold). There was no significant difference between the withaferin A–treated group and the controls (data not shown). Compared with the group treated with cytokine alone, almost all of these genes were significantly downregulated, which indicates a strong anti-inflammatory effect of withaferin A (Fig 2B). To confirm the array results, RT-PCR was repeated using single-primer assays in triplicate. Four genes (RANTES, IP10, MIG, and IL1) that are important in islet cell destruction were selected. iNOS, another gene with equal importance in islet destruction, was also examined. Compared with the cytokine treatment group, withaferin A significantly inhibited expression of all 5 genes (Fig 2C). As a potential anti-inflammatory agent for islet cell transplantation, it was important to ascertain that withaferin A did not affect islet function. In a dose-effect experiment using withaferin A at 0.5, 1, and 2 g/mL, it was observed that after 2 days of culture, 1 g/mL or less did not affect islet viability compared with the control group (68.1% vs 69.4%), whereas a slight decrease was observed with 2 g/mL (50.6% vs 69.4%). Islet potency was tested using a static glucose-stimulated insulin-release assay at 1 g/mL. Stimulation index in the group treated with withaferin A was not significantly different compared with the control
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PENG, OLSEN, TAMURA ET AL
Fig 2. Inhibition of inflammatory gene expression by withaferin A. A, Heat map analysis of inflammatory genes demonstrates that compared with control islets, cytokine-treated islets have significant upregulation of RANTES (CCL5), IP10 (CXCL10), MIG (CXCL9), ITAC (CXCL11), TNF␣, IL1, MCP-2 (CCL8), CXCL5, and IL8. B, Heat map shows the inflammatory genes that were significantly inhibited by withaferin A, 1 g/mL, compared with cytokine-treated islets. C, At single-primer reverse transcriptase-polymerase chain reaction assay, 5 genes (iNOS, IL1, RANTES, MIG, and IP10) were selected, and the level of expression under 3 conditions (control, cytokine/withaferin A–negative, and cytokine/withaferin A-positive) were compared. Fold change analysis demonstrated strong inhibitory effects of withaferin A on inflammatory gene expression.
group (2.65 vs 2.45). Thus, withaferin A did not exert a toxic effect on islet cell function. DISCUSSION
Inflammation is importantly involved in diabetes and islet cell transplantation.3 Cytokine–induced inflammatory gene expression produces detrimental effects on islet cells. In the present study, high expression of RANTES, IP10, and MIG was detected. All are critical for islet cell destruction at immune challenge.7–9 Moreover, cytokine-induced IL1 and iNOS expression can directly trigger beta cell apoptosis.10 However, high Fas expression was not detected in the present study. Our results demonstrate that the naturally occurring anti-inflammatory agent withaferin A inhibits expression of
these inflammatory genes at cytokine stimulation, thus conferring islet cell protection against immune response. To further study the therapeutic potential of withaferin A, in vivo lymphocyte infiltration bioassays and prolongation of islet transplant survival will be tested in a mouse model.
REFERENCES 1. Bennet W, Sundberg S, Groth GC, et al: Incompatibility between human blood and isolated islets of langerhans: a finding with implications for clinical intraportal islet transplantation. Diabetes 48:1907, 1999 2. Merani S, Truong WW, Hancock W, et al: Chemokines and their receptors in islet allograft rejection and as targets for tolerance induction. Cell Transplant 15:295, 2006
WITHAFERIN A INHIBITS ISLET CELL INFLAMMATORY RESPONSE 3. Barshes NR, Wyllie S, Goss JA: Inflammation-mediated dysfunction and apoptosis in pancreatic islet transplantation: implications for intrahepatic grafts. J Leukoc Biol 77:587, 2005 4. Hering BJ, Kandaswamy R, Ansite JD et al: Single-donor, marginal-dose islet transplantation in patients with type 1 diabetes. JAMA 293:830, 2005 5. Kaileh M, Vanden Berghe W, Heyerick A, et al: Withaferin A strongly elicits IkappaB kinase beta hyperphosphorylation concomitant with potent inhibition of its kinase activity. J Biol Chem 282:4253, 2007 6. Matsumoto S, Noguchi H, Naziruddin B, et al: Improvement of pancreatic islet cell isolation for transplantation. Proc (Bayl Univ Med Cent) 20:357, 2007
2061
7. Abdi R, Means TK, Luster AD: Chemokines in islet allograft rejection. Diabetes Metab Res Rev 19:186, 2003 8. Rhode A, Pauza ME, Barral AM, et al: Islet-specific expression of CXCL10 causes spontaneous islet infiltration and accelerates diabetes development. J Immunol 175:3516, 2005 9. Martin AP, Alexander-Brett JM, Canasto-Chibuque C, et al: The chemokine binding protein M3 prevents diabetes induced by multiple low doses of streptozotocin. J Immunol 178:4623, 2007 10. Melloul D. Role of NF-kappaB in beta-cell death. Biochem Soc Trans 36:334, 2008
ABSTRACT Background. After islet cell transplantation, a substantial mass of islets are lost owing to nonspecific inflammatory reactions. Cytokine exposure before or after transplantation can upregulate expression of proinflammatory genes via the nuclear factor-B signaling pathway, eventually resulting in islet loss. Objective. To test the effects of a naturally occurring nuclear factor-B inhibitor, withaferin A, on regulation of inflammatory genes in human islets. Methods. Human pancreatic islets were isolated using a modified Ricordi protocol. Purified islets were cultured for 2 days. The effect of withaferin A treatment on islet cell viability was examined using the fluorescein diacetate-propidium iodide dye exclusion test, and on function using a static glucose stimulation assay. Islet cells were treated with a cytokine mixture (50 U/mL of interleukin-1, 1000 U/mL of tumor necrosis factor-␣, and 1000 U/mL of interferon-␥) for 48 hours with or without withaferin A, 1 g/mL. Treated islets were used for real-time polymerase chain reaction (PCR) array analysis for expression of inflammatory genes, and expression of other selected genes was analyzed using real-time PCR with single primers. Results. Glucose stimulation and viability assays demonstrated that withaferin A was not toxic to islet cells. Of 84 inflammation-related genes examined using real-time PCR array analysis, 9 were significantly upregulated by cytokine treatment compared with the control group. However, addition of withaferin A to the culture significantly inhibited expression of all genes. Conclusion. Withaferin A significantly inhibits the inflammatory response of islet cells with cytokine exposure. ype 1 diabetes mellitus is an autoimmune disease that leads to self-destruction of pancreatic beta cells that produce insulin. Islet cell transplantation to treat brittle forms of diabetes has achieved great success in recent years. However, immediately after transplantation, a substantial amount of islets are lost as a result of the instant bloodmediated inflammatory reaction, an innate immune response characterized by blood coagulation, platelet activation, complement reaction, and cytokine storm.1 In addition, the adaptive immune system slowly destroys the islet graft via lymphocyte infiltration and release of soluble mediators. In both processes, inflammatory cytokines have a crucial role by inducing apoptotic genes and chemokine gene expression in islet cells.2,3 Antibodies and receptor blockers have been used to reduce the effects of inflammatory cyto-
T
kines such as tumor necrosis factor-␣4; however, this strategy cannot entirely block islet graft loss in the long term. Previous studies have demonstrated that nuclear factor-B is the primary transcription factor responsible for expresFrom the Institute of Biomedical Studies, Baylor University, Waco (H.P., B.N.), the Baylor Regional Transplant Institute, Dallas and Fort Worth (G.O., Y.T., M.F.L., B.N.), and Baylor Research Institute, Islet Cell Laboratory, Fort Worth (H.N., S.M.), Texas. This study was supported in part by the Baylor Health Care System Foundation. Address reprint requests to Bashoo Naziruddin, PhD, Islet Cell Laboratory, Transplant Services Department, Baylor University Medical Center, 3500 Gaston Ave, Dallas, TX 75246. E-mail: [email protected]
0041-1345/10/$–see front matter doi:10.1016/j.transproceed.2010.05.131
© 2010 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
2058
Transplantation Proceedings, 42, 2058 –2061 (2010)
WITHAFERIN A INHIBITS ISLET CELL INFLAMMATORY RESPONSE
2059
sion of many inflammatory genes after cytokine stimulation. Withaferin A is a steroidal lactone (Fig 1) derived from Withania somnifera, a plant that has been used for centuries to treat several inflammatory disorders.5 The objective of the present study was to test this naturally occurring antiinflammatory agent for its protective effect on islet cells after cytokine exposure.
transcription–PCR reaction was run using the MX3000 system (Stratagene Corp) with the following program: 95°C for 10 minutes, and 40 cycles of 95°C for 15 seconds and 60°C for 60 seconds. Cycle threshold data were uploaded to a Web-based PCR array data analysis (http://www.sabiosciences.com/pcr/arrayanalysis.php) to analyze the fold change compared with controls:
MATERIALS AND METHODS Islet Isolation
where Ct is cycle threshold, GOI is gene of interest, and HKG is house keeping gene. The procedure for single-primer gene assays was the same except that primers of particular genes other than the entire array plate were used.
Human pancreases designated for research were obtained from normoglycemic cadavers using the 2-layer method. Islets were isolated using a modification of the Ricordi method,6 and stained with dithizone to determine counts and purity. Viability was assessed using the fluorescein diacetate-propidium iodide dye exclusion test. Islets used for the experiments were 89% to 90% viable, with purity of 60% to 85%.
Cytokine and Withaferin A Treatment After isolation, islet cells were treated with a cytokine mixture (50 U/mL of interleukin-1, 1000 U/mL of tumor necrosis factor-␣, and 1000 U/mL of interferon-␥; R&D Systems, Inc, Minneapolis, Minnesota) or cytokine mixture plus withaferin A, 1 g/mL, for 48 hours at 37°C in 5% carbon dioxide (CO2). Nontreated islets were used as controls. All islet cells were cultured in CMRL-based culture medium.
Reverse Transcription–PCR Inflammation Array and Primer Assays After 48 hours islets were harvested and lysed with buffer (Stratagene Corp, La Jolla, California). RNA was purified using an Absolutely RNA Miniprep Kit (Stratagene Corp), and was reverse transcribed into complementary DNA using an RT2 FirstStrand Kit (SABiosciences Corp, Frederick, Maryland). Template cDNA was mixed with RT2 SYBR green/ROX PCR master mix (SABiosciences Corp), and added to each well of a human inflammation gene array plate (SABiosciences Corp). The reverse
Fig 1.
Structure of withaferin A.
Fold change ⫽ 2(⌬Ct [control] ⫺ ⌬Ct [experiment]), ⌬Ct ⫽ Ct (GOI) ⫺ Ct (HKG)
Static Incubation Islet potency was tested using a static glucose-stimulated insulinrelease assay. Islets, 200 IEq, were cultured overnight at 37°C in 5% CO2. Islets in quadruplicates of 50 IEq were stimulated in low-glucose culture medium (2.8 mmol/L) at 37°C in 5% CO2 for 60 minutes. Later, the same islets were stimulated in high-glucose culture medium (20 mmol/L) under the same conditions. Insulin released in both cultures was measured using a human insulin enzyme-linked immunosorbent assay kit (Alpco Diagnostics, Salem, New Hampshire).
RESULTS
Islet cells treated with cytokine or cytokine plus withaferin A, and untreated islet cells were tested for inflammatory gene expression using a real-time PCR array. Of the 84 examined genes, 9 were markedly upregulated compared with untreated islets (Fig 2A): These genes were RANTES (CCL5; 2174-fold), IP10 (CXCL10; 24,255-fold), MIG (CXCL9; 8169-fold), ITAC (CXCL11; 3864-fold), TNF␣(43fold), IL1 (268-fold), MCP-2 (CCL8; 4891-fold), CXCL5 (599-fold), and IL8 (179-fold). There was no significant difference between the withaferin A–treated group and the controls (data not shown). Compared with the group treated with cytokine alone, almost all of these genes were significantly downregulated, which indicates a strong anti-inflammatory effect of withaferin A (Fig 2B). To confirm the array results, RT-PCR was repeated using single-primer assays in triplicate. Four genes (RANTES, IP10, MIG, and IL1) that are important in islet cell destruction were selected. iNOS, another gene with equal importance in islet destruction, was also examined. Compared with the cytokine treatment group, withaferin A significantly inhibited expression of all 5 genes (Fig 2C). As a potential anti-inflammatory agent for islet cell transplantation, it was important to ascertain that withaferin A did not affect islet function. In a dose-effect experiment using withaferin A at 0.5, 1, and 2 g/mL, it was observed that after 2 days of culture, 1 g/mL or less did not affect islet viability compared with the control group (68.1% vs 69.4%), whereas a slight decrease was observed with 2 g/mL (50.6% vs 69.4%). Islet potency was tested using a static glucose-stimulated insulin-release assay at 1 g/mL. Stimulation index in the group treated with withaferin A was not significantly different compared with the control
2060
PENG, OLSEN, TAMURA ET AL
Fig 2. Inhibition of inflammatory gene expression by withaferin A. A, Heat map analysis of inflammatory genes demonstrates that compared with control islets, cytokine-treated islets have significant upregulation of RANTES (CCL5), IP10 (CXCL10), MIG (CXCL9), ITAC (CXCL11), TNF␣, IL1, MCP-2 (CCL8), CXCL5, and IL8. B, Heat map shows the inflammatory genes that were significantly inhibited by withaferin A, 1 g/mL, compared with cytokine-treated islets. C, At single-primer reverse transcriptase-polymerase chain reaction assay, 5 genes (iNOS, IL1, RANTES, MIG, and IP10) were selected, and the level of expression under 3 conditions (control, cytokine/withaferin A–negative, and cytokine/withaferin A-positive) were compared. Fold change analysis demonstrated strong inhibitory effects of withaferin A on inflammatory gene expression.
group (2.65 vs 2.45). Thus, withaferin A did not exert a toxic effect on islet cell function. DISCUSSION
Inflammation is importantly involved in diabetes and islet cell transplantation.3 Cytokine–induced inflammatory gene expression produces detrimental effects on islet cells. In the present study, high expression of RANTES, IP10, and MIG was detected. All are critical for islet cell destruction at immune challenge.7–9 Moreover, cytokine-induced IL1 and iNOS expression can directly trigger beta cell apoptosis.10 However, high Fas expression was not detected in the present study. Our results demonstrate that the naturally occurring anti-inflammatory agent withaferin A inhibits expression of
these inflammatory genes at cytokine stimulation, thus conferring islet cell protection against immune response. To further study the therapeutic potential of withaferin A, in vivo lymphocyte infiltration bioassays and prolongation of islet transplant survival will be tested in a mouse model.
REFERENCES 1. Bennet W, Sundberg S, Groth GC, et al: Incompatibility between human blood and isolated islets of langerhans: a finding with implications for clinical intraportal islet transplantation. Diabetes 48:1907, 1999 2. Merani S, Truong WW, Hancock W, et al: Chemokines and their receptors in islet allograft rejection and as targets for tolerance induction. Cell Transplant 15:295, 2006
WITHAFERIN A INHIBITS ISLET CELL INFLAMMATORY RESPONSE 3. Barshes NR, Wyllie S, Goss JA: Inflammation-mediated dysfunction and apoptosis in pancreatic islet transplantation: implications for intrahepatic grafts. J Leukoc Biol 77:587, 2005 4. Hering BJ, Kandaswamy R, Ansite JD et al: Single-donor, marginal-dose islet transplantation in patients with type 1 diabetes. JAMA 293:830, 2005 5. Kaileh M, Vanden Berghe W, Heyerick A, et al: Withaferin A strongly elicits IkappaB kinase beta hyperphosphorylation concomitant with potent inhibition of its kinase activity. J Biol Chem 282:4253, 2007 6. Matsumoto S, Noguchi H, Naziruddin B, et al: Improvement of pancreatic islet cell isolation for transplantation. Proc (Bayl Univ Med Cent) 20:357, 2007
2061
7. Abdi R, Means TK, Luster AD: Chemokines in islet allograft rejection. Diabetes Metab Res Rev 19:186, 2003 8. Rhode A, Pauza ME, Barral AM, et al: Islet-specific expression of CXCL10 causes spontaneous islet infiltration and accelerates diabetes development. J Immunol 175:3516, 2005 9. Martin AP, Alexander-Brett JM, Canasto-Chibuque C, et al: The chemokine binding protein M3 prevents diabetes induced by multiple low doses of streptozotocin. J Immunol 178:4623, 2007 10. Melloul D. Role of NF-kappaB in beta-cell death. Biochem Soc Trans 36:334, 2008