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Antibody Response to Serpin B13 Induces Adaptive Changes in Mouse Pancreatic Islets and Slows Down the Decline in the Residual Beta Cell Function in Children with Recent Onset of Type 1 Diabetes Mellitus*

Open AccessPublished:November 17, 2015DOI:https://doi.org/10.1074/jbc.M115.687848
      Type 1 diabetes mellitus (T1D) is characterized by a heightened antibody (Ab) response to pancreatic islet self-antigens, which is a biomarker of progressive islet pathology. We recently identified a novel antibody to clade B serpin that reduces islet-associated T cell accumulation and is linked to the delayed onset of T1D. As natural immunity to clade B arises early in life, we hypothesized that it may influence islet development during that time. To test this possibility healthy young Balb/c male mice were injected with serpin B13 mAb or IgG control and examined for the number and cellularity of pancreatic islets by immunofluorescence and FACS. Beta cell proliferation was assessed by measuring nucleotide analog 5-ethynyl-2′-deoxyuridine (5-EdU) incorporation into the DNA and islet Reg gene expression was measured by real time PCR. Human studies involved measuring anti-serpin B13 autoantibodies by Luminex. We found that injecting anti-serpin B13 monoclonal Ab enhanced beta cell proliferation and Reg gene expression, induced the generation of ∼80 pancreatic islets per animal, and ultimately led to increase in the beta cell mass. These findings are relevant to human T1D because our analysis of subjects just diagnosed with T1D revealed an association between baseline anti-serpin activity and slower residual beta cell function decline in the first year after the onset of diabetes. Our findings reveal a new role for the anti-serpin immunological response in promoting adaptive changes in the endocrine pancreas and suggests that enhancement of this response could potentially help impede the progression of T1D in humans.

      Introduction

      Type 1 diabetes mellitus (T1D)
      The abbreviations used are: T1D, type 1 diabetes mellitus; NOD, nonobese diabetic; STZ, streptozotocin; IF, immunofluorescence; 5-EdU, 5-ethynyl-2′-deoxyuridine.
      is an autoimmune condition that affects people of all ages. Although T1D was traditionally considered a pediatric disease, it has become clear that individuals over 20 years old can also develop detectable autoimmunity against pancreatic islets with concomitant sudden onset of insulin dependence (
      • Karjalainen J.
      • Salmela P.
      • Ilonen J.
      • Surcel H.M.
      • Knip M.
      Comparison of childhood and adult type I diabetes mellitus.
      ). To date, the modifying factors that precipitate the clinical manifestation of autoimmune diabetes at different ages are largely unknown.
      We have focused on factors that regulate the timing of clinical onset of T1D. Recent observations from our laboratory have revealed a novel autoantibody directed against the serpin B13 protease inhibitor (
      • Abts H.F.
      • Welss T.
      • Mirmohammadsadegh A.
      • Köhrer K.
      • Michel G.
      • Ruzicka T.
      Cloning and characterization of hurpin (protease inhibitor 13): a new skin-specific, UV- repressible serine proteinase inhibitor of the ovalbumin serpin family.
      ,
      • Welss T.
      • Sun J.
      • Irving J.A.
      • Blum R.
      • Smith A.I.
      • Whisstock J.C.
      • Pike R.N.
      • von Mikecz A.
      • Ruzicka T.
      • Bird P.I.
      • Abts H.F.
      Hurpin is a selective inhibitor of lysosomal cathepsin L and protects keratinocytes from ultraviolet induced apoptosis.
      ) and demonstrated that it partially protects against early onset autoimmune diabetes (
      • Czyzyk J.
      • Henegariu O.
      • Preston-Hurlburt P.
      • Baldzizhar R.
      • Fedorchuk C.
      • Esplugues E.
      • Bottomly K.
      • Gorus F.K.
      • Herold K.
      • Flavell R.A.
      Enhanced anti-serpin antibody activity inhibits autoimmune inflammation in type 1 diabetes.
      ). In T1D susceptible nonobese diabetic (NOD) mice, elevated secretion of anti-serpin B13 autoantibody is associated with protection from diabetes before 16 weeks of age, whereas decreased secretion of this antibody (Ab) in humans is associated with T1D onset before age 5 years (
      • Czyzyk J.
      • Henegariu O.
      • Preston-Hurlburt P.
      • Baldzizhar R.
      • Fedorchuk C.
      • Esplugues E.
      • Bottomly K.
      • Gorus F.K.
      • Herold K.
      • Flavell R.A.
      Enhanced anti-serpin antibody activity inhibits autoimmune inflammation in type 1 diabetes.
      ). These observations suggest an inverse relationship between the serpin B13 autoantibody response and the appearance of the clinical features of T1D. We further linked the serpin Ab to reduced autoimmune inflammation in pancreatic islets, likely due to enhanced extracellular cleavage of key cell surface molecules expressed in T and B cells (
      • Baldzizhar R.
      • Fedorchuk C.
      • Jha M.
      • Rathinam C.
      • Henegariu O.
      • Czyzyk J.
      Anti-serpin antibody-mediated regulation of proteases in autoimmune diabetes.
      ). Of note, serpins have been implicated as anti-apoptotic agents that improve islet survival (
      • Millet I.
      • Wong F.S.
      • Gurr W.
      • Wen L.
      • Zawalich W.
      • Green E.A.
      • Flavell R.A.
      • Sherwin R.S.
      Targeted expression of the anti-apoptotic gene CrmA to NOD pancreatic islets protects from autoimmune diabetes.
      ,
      • Zhang B.
      • Lu Y.
      • Campbell-Thompson M.
      • Spencer T.
      • Wasserfall C.
      • Atkinson M.
      • Song S.
      α1-Antitrypsin protects beta-cells from apoptosis.
      ), although in a model of prevention of autoimmune diabetes by limited apoptosis, transgenic intraislet expression of serpin-like protein CrmA reduces rather than enhances preventive effect (
      • Hugues S.
      • Mougneau E.
      • Ferlin W.
      • Jeske D.
      • Hofman P.
      • Homann D.
      • Beaudoin L.
      • Schrike C.
      • Von Herrath M.
      • Lehuen A.
      • Glaichenhaus N.
      Tolerance to islet antigens and prevention from diabetes induced by limited apoptosis of pancreatic beta cells.
      ).
      Notwithstanding the anti-inflammatory impact of serpin B13 Ab, our previous studies suggested that an additional mechanism may account for the protective effect of this immunological response in the setting of T1D. First, we found that the natural Ab response to serpin B13 arises early in life but declines in 8- to 12-week-old mice and the first 5 to 10 years in humans (
      • Czyzyk J.
      • Henegariu O.
      • Preston-Hurlburt P.
      • Baldzizhar R.
      • Fedorchuk C.
      • Esplugues E.
      • Bottomly K.
      • Gorus F.K.
      • Herold K.
      • Flavell R.A.
      Enhanced anti-serpin antibody activity inhibits autoimmune inflammation in type 1 diabetes.
      ). This early transient Ab response to serpin B13 suggests that anti-serpin autoantibodies have limited, if any, effect on intraislet inflammation that develops after early childhood. Rather serpin Ab may affect cell growth and/or death in non-inflamed islets before autoimmunity strikes, ultimately protecting endocrine pancreatic tissue. The published reports on the enhanced regenerative potential of pancreatic tissue at a young age lend support to this possibility (
      • Kassem S.A.
      • Ariel I.
      • Thornton P.S.
      • Scheimberg I.
      • Glaser B.
      Beta-cell proliferation and apoptosis in the developing normal human pancreas and in hyperinsulinism of infancy.
      • Meier J.J.
      • Butler A.E.
      • Saisho Y.
      • Monchamp T.
      • Galasso R.
      • Bhushan A.
      • Rizza R.A.
      • Butler P.C.
      Beta-cell replication is the primary mechanism subserving the postnatal expansion of beta-cell mass in humans.
      ,
      • Perl S.
      • Kushner J.A.
      • Buchholz B.A.
      • Meeker A.K.
      • Stein G.M.
      • Hsieh M.
      • Kirby M.
      • Pechhold S.
      • Liu E.H.
      • Harlan D.M.
      • Tisdale J.F.
      Significant human beta-cell turnover is limited to the first three decades of life as determined by in vivo thymidine analog incorporation and radiocarbon dating.
      • Finegood D.T.
      • Scaglia L.
      • Bonner-Weir S.
      Dynamics of beta-cell mass in the growing rat pancreas: estimation with a simple mathematical model.
      ). Second, our previous finding of serpin B13 expression in pancreatic exocrine ducts suggests that the immune response to this molecule may regulate the relationship between distinct pancreatic tissue compartments. In this hypothetical scenario, a ductal immunological response to serpin B13 could intensify functional cross-talk with the endocrine tissue and facilitate the renewal of insulin-producing cells. Research by other groups also hints at the possibility of deriving beta cells and their progenitors from pancreatic ductal cells or adjacent regions, at least in the setting of acute injury in animal models (
      • Wang R.N.
      • Klöppel G.
      • Bouwens L.
      Duct- to islet-cell differentiation and islet growth in the pancreas of duct ligated adult rats.
      ,
      • Bonner-Weir S.
      • Baxter L.A.
      • Schuppin G.T.
      • Smith F.E.
      A second pathway for regeneration of adult exocrine and endocrine pancreas: a possible recapitulation of embryonic development.
      ) or chronic human pancreatic disease (
      • Jones L.C.
      • Clark A.
      Beta-cell neogenesis in type 2 diabetes.
      ,
      • Phillips J.M.
      • O'Reilly L.
      • Bland C.
      • Foulis A.K.
      • Cooke A.
      Patients with chronic pancreatitis have islet progenitor cells in their ducts, but reversal of overt diabetes in NOD mice by anti-CD3 shows no evidence for islet regeneration.
      ).
      The present study is the first attempt to elucidate serpin Ab-mediated regulation of endocrine islet cells under normal conditions. To ensure clinical relevancy, we employed a monoclonal Ab (mAb) as an enhancing agent and applied it to the animals in the third week of life when the natural response to serpin B13 is at its maximum. We found that both the number of pancreatic islets and beta cell proliferation are significantly increased after exposure to serpin mAb. Moreover, to translate our findings to human we examined the impact of serpin B13 Abs in children in the first year after the onset of T1D and found that this Ab response is associated with slower progression of beta cell defect. Together with our previous reports, these observations have implications for a better understanding of how anti-serpin activity may fine-tune the balance between the destructive forces of autoimmune inflammation and adaptive changes in the islets, and point to this activity as a potential modifying factor of the natural history of diabetes.

      Discussion

      Serpin protease inhibitors play a fundamental role in preserving the structural and functional integrity of cells and tissues in the body (
      • Irving J.A.
      • Pike R.N.
      • Lesk A.M.
      • Whisstock J.C.
      Phylogeny of the serpin superfamily: implications of patterns of amino acid conservation for structure and function.
      ,
      • Luke C.J.
      • Pak S. C
      • Askew Y. S
      • Naviglia T.L.
      • Askew D.J.
      • Nobar S.M.
      • Vetica A.C.
      • Long O.S.
      • Watkins S.C.
      • Stolz D.B.
      • Barstead R.J.
      • Moulder G.L.
      • Brömme D.
      • Silverman G.A.
      An intracellular serpin regulates necrosis by inhibiting the induction and sequelae of lysosomal injury.
      ). Autoantibody-mediated dysregulation of these functions can have serious clinical complications (
      • Jackson J.
      • Sim R.B.
      • Whelan A.
      • Feighery C.
      An IgG autoantibody which inactivates C1-inhibitor.
      ,
      • Cacoub P.
      • Frémeaux-Bacchi V.
      • De Lacroix I.
      • Guillien F.
      • Kahn M.F.
      • Kazatchkine M.D.
      • Godeau P.
      • Piette J.C.
      A new type of acquired C1 inhibitor deficiency associated with systemic lupus erythematosus.
      ). In contrast to other autoimmune conditions where the presence of serpin autoantibodies is indicative of disease, we found that the presence of the immunoglobulin response to serpin B13 is protective in T1D (
      • Czyzyk J.
      • Henegariu O.
      • Preston-Hurlburt P.
      • Baldzizhar R.
      • Fedorchuk C.
      • Esplugues E.
      • Bottomly K.
      • Gorus F.K.
      • Herold K.
      • Flavell R.A.
      Enhanced anti-serpin antibody activity inhibits autoimmune inflammation in type 1 diabetes.
      ). Our previous results demonstrated that serpin B13 Abs reduce lymphocytic infiltration of pancreatic islets (
      • Czyzyk J.
      • Henegariu O.
      • Preston-Hurlburt P.
      • Baldzizhar R.
      • Fedorchuk C.
      • Esplugues E.
      • Bottomly K.
      • Gorus F.K.
      • Herold K.
      • Flavell R.A.
      Enhanced anti-serpin antibody activity inhibits autoimmune inflammation in type 1 diabetes.
      ,
      • Baldzizhar R.
      • Fedorchuk C.
      • Jha M.
      • Rathinam C.
      • Henegariu O.
      • Czyzyk J.
      Anti-serpin antibody-mediated regulation of proteases in autoimmune diabetes.
      ). In the present study, we found that serpin B13 can also influence adaptive changes in pancreatic tissue by increasing output of small islets soon after exposure to serpin Ab, and then during the following months, causing a similar increase in the number of larger islets.
      To explain these adaptive changes, we focused on the impact of serpin B13 mAb on beta cells proliferation, reasoning that the newly formed islets could represent preexisting tiny clusters of replicating beta cells. Total beta hypercellularity also implied proliferation as the mechanism of expansion. Indeed, quantification of cells positive for both 5-EdU and insulin revealed that anti-serpin activity enhanced beta cell proliferation, especially in small pancreatic islets. Our discovery of high Reg gene expression in pancreatic islets lends further support to the hypothesis that enhanced humoral immunity to serpin molecules can stimulate adaptive changes because Reg proteins have been implicated in the processes of islet proliferation and regeneration. For example, Reg1 is important in islet cell proliferation, and its expression is increased during islet regeneration (
      • Terazono K.
      • Yamamoto H.
      • Takasawa S.
      • Shiga K.
      • Yonemura Y.
      • Tochino Y.
      • Okamoto H.
      A novel gene activated in regenerating islets.
      ), whereas Reg3δ has been shown to stimulate the differentiation of putative pancreatic stem cells in diabetes (
      • Okamoto H.
      The Reg gene family and Reg proteins: with special attention to the regeneration of pancreatic beta-cells.
      ).
      Although our results are somewhat expected because we used young animals with relatively well preserved beta cell proliferation (
      • Kushner J.A.
      The role of aging upon β cell turnover.
      ) it is possible that an immune response to serpin molecules may influence the endocrine cell adaptation by additional mechanisms. Indeed, observed by us relatively modest up-regulation in beta cell proliferation may not fully account for noticeable changes in islet numbers and their cellularity following injection of serpin B13 mAb. Moreover, we previously reported that the target of our serpin Ab is expressed in pancreatic ducts (
      • Baldzizhar R.
      • Fedorchuk C.
      • Jha M.
      • Rathinam C.
      • Henegariu O.
      • Czyzyk J.
      Anti-serpin antibody-mediated regulation of proteases in autoimmune diabetes.
      ). In addition, others have demonstrated that ductal epithelium or an adjacent niche plays a role in endocrine progenitor cell activation (
      • Xu X.
      • D'Hoker J.
      • Stangé G.
      • Bonné S.
      • De Leu N.
      • Xiao X.
      • Van de Casteele M.
      • Mellitzer G.
      • Ling Z.
      • Pipeleers D.
      • Bouwens L.
      • Scharfmann R.
      • Gradwohl G.
      • Heimberg H.
      Beta cells can be generated from endogenous progenitors in injured adult mouse pancreas.
      ,
      • Wu F.
      • Guo L.
      • Jakubowski A.
      • Su L.
      • Li W.C.
      • Bonner-Weir S.
      • Burkly L.C.
      TNF-like weak inducer of apoptosis (TWEAK) promotes beta cell neogenesis from pancreatic ductal epithelium in adult mice.
      ). All these observations suggest that de novo beta cell formation may occur following ductal stimulation with serpin antibodies. However, the proposal that pancreatic exocrine ducts are a source of beta cells in animals exposed to serpin antibody has not been directly assessed in this study. Such investigations should employ genetic lineage tracing of ductal cells in mice with an inducible Cre recombinase-driven promoter that can act as a duct marker (
      • Solar M.
      • Cardalda C.
      • Houbracken I.
      • Martín M.
      • Maestro M.A.
      • De Medts N.
      • Xu X.
      • Grau V.
      • Heimberg H.
      • Bouwens L.
      • Ferrer J.
      Pancreatic exocrine duct cells give rise to insulin-producing β cells during embryogenesis but not after birth.
      ,
      • Inada A.
      • Nienaber C.
      • Katsuta H.
      • Fujitani Y.
      • Levine J.
      • Morita R.
      • Sharma A.
      • Bonner-Weir S.
      Carbonic anhydrase II-positive pancreatic cells are progenitors for both endocrine and exocrine pancreas after birth.
      ).
      The exact role of increased numbers of small islets in our system remains to be elucidated. Small islets that emerge after exposure to serpin Abs could represent a dormant pool that can be activated during increased metabolic demand (
      • Weir G.C.
      • Bonner-Weir S.
      Sleeping islets and the relationship between β-cell mass and function.
      ). Because pancreatic endocrine cells are thought to have a long lifespan (
      • Cnop M.
      • Hughes S.J.
      • Igoillo-Esteve M.
      • Hoppa M.B.
      • Sayyed F.
      • van de Laar L.
      • Gunter J.H.
      • de Koning E.J.
      • Walls G.V.
      • Gray D.W.
      • Johnson P.R.
      • Hansen B.C.
      • Morris J.F.
      • Pipeleers-Marichal M.
      • Cnop I.
      • Clark A.
      The long lifespan and low turnover of human islet beta cells estimated by mathematical modelling of lipofuscin accumulation.
      ), it is possible that the newly formed islets that arise in young mice following serpin B13 Ab injection can survive for a considerable period of time and are responsible for further beta cell and islet expansion that was noted in our study (Fig. 1F). A related question is how late can serpin B13 mAb treatment induce adaptive changes in islets? Although our serpin B13 mAb treatment coincided with the second wave of natural islet neogenesis that occurs between the second and third week of life in rodents (
      • Finegood D.T.
      • Scaglia L.
      • Bonner-Weir S.
      Dynamics of beta-cell mass in the growing rat pancreas: estimation with a simple mathematical model.
      ), it is equally important to know whether a similar treatment of aged mice (e.g. >1 year old) can overcome their severely compromised islet regenerative potential (
      • Teta M.
      • Long S.Y.
      • Wartschow L.M.
      • Rankin M.M.
      • Kushner J.A.
      Very slow turnover of beta-cells in aged adult mice.
      ,
      • Saisho Y.
      • Butler A. E
      • Manesso E.
      • Elashoff D.
      • Rizza R.A.
      • Butler P.C.
      β-Cell mass and turnover in humans: effects of obesity and aging.
      ) and up-regulate the number of islets or induce other islet adaptive changes.
      The studies of NOD mice expressing high levels of serpin B13 autoantibodies indicated an almost 50% increase in the number of pancreatic islets, although this value in Balb/c mice that received serpin B13 mAb was only 20%. Although mouse strain difference could account for partial resistance to serpin B13 mAb, other reasons should also be considered. For example, the limited response to our mAb may reflect a weak effect of this antibody binding on serpin B13 conformation. A passive immunization with a mAb that interacts with a critical fragment of serpin B13 (e.g. the hinge region in the reactive site loop that allows serpins to neutralize target proteases (2 and 3)) is an alternative that should be tested. Notably, this strategy to influence protease activity is in accordance with our earlier report that Ab serpin B13 reduces autoimmune inflammation in T1D, likely through enhanced cleavage of key cell-surface molecules on lymphocytes residing in pancreatic islets (
      • Baldzizhar R.
      • Fedorchuk C.
      • Jha M.
      • Rathinam C.
      • Henegariu O.
      • Czyzyk J.
      Anti-serpin antibody-mediated regulation of proteases in autoimmune diabetes.
      ). However, it should be stressed that the role of proteases has not been addressed in this study, and whether proteolytic activity contributes to islet adaptive changes remains to be determined.
      The translational aspect of our project suggests that monitoring anti-serpin activity in humans helps predict progression of changes in the well being of beta cells throughout the first year of T1D. This conclusion is supported by examination of both C-peptide secretion profiles and IDAA1C levels as a surrogate measure. However, it is unclear why only patients with weak (+), but not strong (++) secretion of serpin B13 Ab demonstrated significantly higher stimulated C-peptide response compared with negative controls at the 6-month follow up. One possible interpretation of this finding could be the potentially different kinetics of immunological responses to serpin molecules. More specifically, weakly positive Ab at baseline may represent a declining response that was at its maximum long before the onset of diabetes. Consequently, patients with long-lasting immunity to serpin molecules would have pancreatic islets better prepared to resist their final destruction compared with other patients. By the same token, a strong presence of serpin Ab at baseline may reflect a more recent immunological response, thereby offering the patients little opportunity to develop islet adaptive changes immediately after diabetes onset. In future, comparing anti-serpin activity in at-risk progressors versus non-progressors, or following this activity in subjects with ongoing diabetes for 2 to 5 years after disease onset, would give us a better insight into the long-term impact of serpin Ab on diabetes. Addressing this and other questions will require examinations of serum samples from well defined subject cohorts and should be attainable through continued collaboration with other groups that possess these precious collections of human specimens.

      Author Contributions

      Y. K. and C.-W. L. contributed equally to this work. Y. K. and C.-W. L. contributed to study design, researched and analyzed the data, and reviewed and edited the manuscript. E. M. and R. B. researched the data. N. J. contributed to study design, and reviewed and edited the manuscript. J. C. designed the experiments, analyzed the data and wrote the manuscript.

      Acknowledgments

      We thank Sarah Muller and the Type 1 Diabetes TrialNet for help in providing human serum samples and supporting data. We also thank Dr. Jerry Palmer for coordinating review of our manuscript by the TrialNet Publications and Presentations Subcommittee prior to its submission for publication.

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