Institute of Food Sciences, CNR Avellino, Italy Therapeutical implication of regulatory cells and cytokines in celiac disease Carmen Gianfrani Mastering the coeliac condition: from medicine to social sciences and food technology Florence, Congress Palace, 29 31 March 2012
Oral Tolerance In healthy jejunal mucosa food antigens and commensal flora are beneficial and tolerated food antigens commensal bacteria Mowat, Nat Rev Immunol 2003 Oral Tolerance is a tightly controlled immune phenomenon. The intestinal immune system has to discriminate between harmful and beneficial luminal antigens Key role of Regulatory T cells and cytokines IL 10 and TGFβ in OT Weiner Immunol Rev. 2011 Powrie, Immunol Rev. 2006
Abnormal immune response to gluten occurs in in celiac mucosa but not in healthy gut 7000 6000 IFN-γ SFC/1x10 6 cells 5000 4000 3000 2000 1000 Gianfrani, J Immunol 2003 Gianfrani, J Immunol 2006 Mazzarella, Gastroenterology 2008 0 non-cd CD Lundin, J Exp Med 1993 Molberg, Scand J Immunol 2006 Is oral tolerance broken in CD?
Both pro inflammatory and regulatory cytokines are upregulated in celiac mucosa
T REGULATORY (Tr) CELLS CD4-8 - CD4 + NK-T CD8 + Tr1 cells (IL-10 +, TGF-beta +, IFN- +, IL-4 -, IL-2 low/- ) Th3 cells (IL-10+, TGF-beta+) CD4+CD25+Foxp3+ (IL-10+, TGF-beta+)
IL 10 and TGF beta are key cytokines in controlling immune response to gluten in celiacs 60 3-H-TdR (cpmx10 3 ) 50 40 30 20 10 IFN (pg/ml) 1400 1200 1000 800 600 400 200 0 0 cells gliadin gliadin + ail10r gliadin + atgf gliadin + both cells + ail10r cells + atgf cells gliadin gliadin + ail10r gliadin + atgf gliadin + both cells + ail10r cells + atgf Gianfrani C. et al J Immunol 2006
IL 10 and TGF β control T cell response to gliadin in children at high risk to develop CD (PreventCD EU FP6 2005 FOOD4B) IFN-γ (Fold Increase) 35 30 25 20 15 10 PrCD with atrophic mucosa N=6 P = 0.05 35 30 25 20 15 10 PrCD with normal mucosa N=6 p=0.026 5 5 0 gliadin gliadin + a-il-10r + a-tgfbeta gliadin+tg gliadin+tg + a-il-10r + a-tgfbeta 0 gliadin gliadin + a-il-10r + a-tgfbeta gliadin+tg gliadin+tg + a-il-10r + a-tgfbeta Gianfrani et al unpublished see poster #26 for details
Immune response to gliadin at early CD state PreventCD EU FP6 2005 FOOD4B) PrCD 102-093 AGA ttg(iga) Marsh grade CD3 γδ CD25 IgA deposit 40,2 0,1 M1 36 2,3 5 Absent 700 600 IFN- (pg/ml) 500 400 300 200 100 0 medium gliadin gliadin gliadin+tg + a-il-10r + a-tgfbeta gliadin+tg + a-il-10r + a-tgfbeta First biopsy: normal mucosa Gianfrani et al unpublished see poster #26 for details Second biopsy after 30 months: atrophy, ttg 100U/ml
IL 10 and TGFβ producing Treg (Tr1) cells are present in celiac mucosa and suppress immune response to gliadin IL-10 net pg/ml 1200 1000 800 600 400 200 0 Tr1.7 Tr1 phenotype CD3+CD4+ TCR a/b+ B7 integrin+ CD103 neg CD45RO+ 11.000 21000 TCC Tr1.9 IL-4 TGF- IL-2 -IFN 7 Stimulation index 6 5 4 3 2 1 0 Th.6 (R) Tr1.7 (S) R:5x10 4 S:5x10 4 S:R 2:1 S:R 1:1 11 S:R 0.2:1 15 S:R 0.1:1 58 70 R + S R +R Gianfrani C. et al J Immunol 2006
No IL 10 and TGFβ producing Treg (Tr1) cells in nonceliac, healthy subjects IFN- -SFC/1x10 6 cells 12000 10000 8000 6000 4000 gliadin gliadin + -IL-10R and -TGF PHA 2000 0 T1D281004 T1D031104 T1D141205 T1D200406 T1D061207 Gianfrani C. et al J Immunol 2006 Gianfrani C unpublished
No defect in the presence and suppressive function of IL 10 and TGFβ T regulatory cells in CD No evidence of Tr1 cell reactive to gliadin in healthy mucosa In DQ2 Tg mice tolerance to deamidated gliadin is mantained by regulatory IL 10+Tr1 cells (Du Pre MF Gastroenterology 2011)
Increased CD4+Foxp3+ T regs in celiac mucosa Control mucosa Untreated mucosa Foxp3+; CD4+ Zanzi, Am J Gastr 2011
Intraepithelial CD8 + cells have a regulatory function in CD Baghat et al, J Clin Invest 2008
No defect in the presence and suppressive function of CD4+CD25+Foxp3+ and CD8+TCRγδ+ Treg In normal condition gluten seems to be ignored by the gut immune system
Standpoint: activation of both pro inflammatory and regulatory pathways in CD mucosa gluten gluten Regulatory Immune response IL10+TGF + Tr1 CD4+CD25+Foxp3+ Inflammatory Response Immune Regulation Treg based therapy
Very low rate of in vitro expansion of gliadin reactive Treg from biopsy Gliadin (± ttg) Biopsy (3-5 mm) INF γ, IL 2, IL 21 Proliferation Gliadin-Th cells Gianfrani, Gastroenterology 2007 Camarca, J Immunol 2009 Rizzello, App Env Microb 2007 Bergamo, Mol Nutr Food Res. 2011 Maglio, Scand J Gastroenterol. 2011 Gliadin-Treg cells IL 10, TGFbeta Proliferation
Gliadin IL 10+ T cells in the blood of celiac and not in healthy donors IL-10-SFC/1x10 6 PBMC 9000.00 8000.00 7000.00 6000.00 5000.00 4000.00 3000.00 p<0.009 ns p<0.0006 Control donors Celiacs 2000.00 1000.00 0.00 gliadin gliadin-tg LPS PHA Camarca et al unpublished
Is Treg based therapy suitable in celiac disease? Possible strategies: i. In vitro induction/expansion of gut homing Tr1 and delivery into patients. ii. Orally administration of genetically modified bacteria (IL 10/gliadin secreting lactococcus lactis), to in vivo induce or expand Tr1 cells iii. Gluten peptide vaccination: in vivo induction of specific IL 10+Tr1 cells?
Adoptive Transfer of Treg in Clinical Trials Reviewed by Gregori S. Frontiers in Immunology. Feb 2012
Contributes Institute of Food Sciences, CNR, Avellino, Italy Carmen Gianfrani Giuseppe Mazzarella Alessandra Camarca Stefania Picascia Rosita Stefanile Department of Pediatrics University Federico II, Naples, Italy Salvatore Auricchio Riccardo Troncone Luigi Greco Virginia Salvati Delia Zanzi Thanks to AIC & Celiac People