Abstract
Antiinflammatory Effects of Withaferin A in Islet Transplantation and Pancreatitis
Mazhar A. Kanak, Ph.D.
Mentor: Bashoo Naziruddin, Ph.D.
Islet transplantation is a promising treatment for patients severely affected by type
1 diabetes and chronic pancreatitis. Islets are transplanted into the portal vein of the liver, and direct contact of islets with blood initiates a strong innate immune response called instant blood-mediated inflammation response (IBMIR). Approximately 50% to 60% of islets may be lost during the peritransplant period due to IBMIR. A transcription factor known as nuclear factor kappa B (NFκB) primarily mediates the proinflammatory response in islets. Preliminary studies using withaferin A (WA), a plant-derived inhibitor of NFκB, showed protection of islets from proinflammatory cytokine-mediated damage.
An in vitro tube model for IBMIR was developed to determine the inhibitory effect of
WA. After transplantation, islet damage was evaluated using C-peptide and proinsulin levels in serum, which are influenced by metabolic stimuli. Identification of a biomarker specific for islet damage uninfluenced by
physiological changes is a critical need. MicroRNA 375 is highly expressed in β cells of
islets, and damage of islets results in release of this miRNA into the bloodstream.
Analysis of serum samples during and after islet infusion revealed high levels of
microRNA 375, indicating damage by IBMIR, and the estimated islet loss was
comparable to that in previous reports. Serum microRNA 375 was also analyzed to
validate the inhibitory effect of WA on IBMIR in vitro.
Chronic pancreatitis is an inflammatory disease affecting the exocrine and
subsequently endocrine function of the pancreas. Currently, there is no therapeutic
approach to delay progression of the disease. The underlying mechanism of pathogenesis
of chronic pancreatitis is not clearly understood. The activation of NFκB in acinar cells
has been reported to enhance the severity of chronic pancreatitis. NFκB inhibition by WA
was tested in a cerulein-induced pancreatitis mouse model. The sustained inflammation
that leads to chronic pancreatitis is potentiated by endoplasmic reticulum stress and inflammasome activation within the pancreas. Treatment with WA prevented activation of these mechanisms, as evidenced by molecular analysis and histological assessment.
In summary, inhibition of NFκB by WA could be a promising strategy to treat chronic pancreatic diseases. Antiinflammatory Effects of Withaferin A in Islet Transplantation and Pancreatitis
by
Mazhar A. Kanak B. Tech., M.S.
A Dissertation
Approved by the Institute of Biomedical Studies
Robert Kane, Ph.D., Chairperson
Submitted to the Graduate Faculty of Baylor University in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy
Approved by the Dissertation Committee
Bashoo Naziruddin, Ph.D., Chairperson
Robert Kane, Ph.D.
Sangkon Oh, Ph.D.
Christopher Kearney, Ph.D.
Michael Trakselis, Ph.D.
Michael C. Lawrence, Ph.D.
Accepted by the Graduate School August 2015
J. Larry Lyon, Ph.D., Dean
Page bearing signatures is kept on file in the Graduate School. Copyright © 2015 by Mazhar A. Kanak
All rights reserved TABLE OF CONTENTS
LIST OF FIGURES……………………………………………………………………………..viii
LIST OF TABLES………………………………………………………………………………...x
LIST OF ABBREVIATIONS………………………………………………………………….....xi
ACKNOWLEDGMENTS……………………………………………………………………....xvi
DEDICATION…………………………………………………………………………………xviii
CHAPTER ONE…………………………………………………………………………………..1
Introduction………………………………………………………………………………..1
Diseases of the Pancreas…………………………………………………………...2
Islet Transplantation……………………………………………………………….4
Causes of the Inflammatory Response in Islet Transplantation……………………4
Pancreas Donors…………………………………………………………………...6
Ischemic Time……………………………………………………………………..6
Islet Isolation Process……………………………………………………………...7
Pretransplant Islet Culture…………………………………………………………8
Peritransplant Inflammation: IBMIR……………………………………………...9
Posttransplant Inflammation……………………………………………………..19
Inflammatory Response from Islets……………………………………………...21
Antiinflammatory Strategies to Improve Islet Transplantation…………………..22
Withaferin A……………………………………………………………………..26
Future Directions…………………………………………………………………29
v CHAPTER TWO………………………………………………………………………………...31
Alleviation of Instant Blood-Mediated Inflammatory Reaction in Autologous Conditions through Treatment of Human Islets with Nuclear Factor-Kappa B Inhibitors…………………………………………………………………………………31
Introduction……………………………………………………………………....31
Materials and Methods…………………………………………………………...33
Results……………………………………………………………………………36
Discussion………………………………………………………………………..41
CHAPTER THREE……………………………………………………………………………...47
Evaluation of MicroRNA 375 as a Novel Biomarker for Graft Damage in Clinical Islet Transplantation…………………………………………………………….47
Introduction………………………………………………………………………47
Materials and Methods…………………………………………………………...49
Results……………………………………………………………………………51
Discussion………………………………………………………………………..58
CHAPTER FOUR………………………………………………………………………………..63
Delineating the Molecular Pathogenesis of Chronic Pancreatitis and Use of NFκB Inhibitor to Prevent its Progression……………………………………………….63
Introduction……………………………………………………………………....63
Materials and Methods…………………………………………………………...65
Results……………………………………………………………………………70
Discussion………………………………………………………………………..85
CHAPTER FIVE………………………………………………………………………………...92
Summary and Conclusions………………………………………………………………92
Summary…………………………………………………………………………92
vi Alleviation of Instant Blood Mediated Inflammatory Reaction in Autologous Conditions through Treatment of Human Islets with NFκB inhibitors………………………………………………………………….93
Evaluation of miRNA 375 as a Novel Biomarker for Graft Damage In Clinical Islet Transplantation………………………………………………….95
Delineating the Molecular Pathogenesis of Chronic Pancreatitis and Use of NFκB Inhibitor to Prevent its Progression…………………………….....97
Conclusions………………………………………………………………………99
Bibliography……………………………………………………………………………100
vii LIST OF FIGURES
Figure Page
1.1 Factors that induce an inflammatory reaction to islet grafts………………………………5
1.2 Flow diagram for method of miniaturized tube model……………………………………11
1.3 Autologous IBMIR activation in vitro and validation parameters……………………….11
1.4 Elevation of pro-inflammatory cytokines/chemokines in serum after IBMIR……………12
1.5 Activation of IBMIR in clinical TPIAT patient samples………………………………....13
1.6 Hypothetical Model of the mechanism of IBMIR………………………………………..14
1.7 WA improved syngeneic mice islet transplant outcome and reduced cytokine induced apoptosis………………………………………………………………………...27
1.8 Western blot showing inhibition of NFκB activation by WA…………………………….28
1.9 WA inhibits proinflammatory cytokine/chemokine production by islet treated with cytokines……………………………………………………………………………28
2.1 Viability of islets after culture in autologous blood……………………………………....36
2.2 Autologous islet damage and the impact of NFκB inhibitors…………………………….37
2.3 Inhibition of proinflammatory cytokine secretion by NFκB inhibitors…………………..39
2.4 Neutrophil infiltration in islet clots blocked by NFκB inhibitors………………………...40
2.5 Tissue factor (TF) expression in islets after exposure to autologous blood………………41
3.1 miR375 expression correlates with islet mass and increases similarly to C-peptide in response to islet damage in a miniaturized in vitro tube model……………………….53
3.2 WA blocked islet damage by IBMIR: miRNA 375, c-peptide and proinsulin…………..55
3.3 miRNA levels in vivo indicate immediate islet damage after infusion…………………..56
3.4 Standard curve plot for the quantification of miRNA375……………………………….57
viii 3.5 Correlation of miRNA 375 levels and IEQ transplanted………………………………...57
4.1 Protocol for development and treatment of chronic pancreatitis………………………...67
4.2 WA reduces severity of AP and abolishes immune cell infiltration……………………..72
4.3 WA can prevent development of Chronic Pancreatitis (CP)….…………………………76
4.4 Withaferin A (WA) can abrogate progression of severe chronic pancreatitis (CP)- therapeutic model………………………………………………………………………...77
4.5 WA reduces cerulein induced acinar damage and blocks neutrophil infiltration………..78
4.6 WA inhibits cerulein induced NFκB in acinar cells and downregulates expression of proinflammatory and pro-apoptotic molecules………………………………………..80
4.7 Withaferin A (WA) downregulates cerulein-induced expression of proinflammatory and proapoptotic molecules in therapeutic CP model…………………………………....81
4.8 WA reduces endoplasmic reticulum (ER) stress……………...…………………………83
4.9 WA reduces expression of inflammasome related genes……………….…………….....84
4.10 Real-time PCR analysis of ER stress and inflammasome related genes in therapeutic model………………………………………………………………….……..86
4.11 Heat map of proinflammatory and proapoptotic genes in healthy and CP patients..…….87
4.12 Schematic representation of the signaling pathways operative in the incidence of CP and the blockade by WA……………………………………………………………...... 91
ix LIST OF TABLES
Table Page
1.1 Antiinflammatory agents in clinical allogeneic islet transplantation…………………….25
3.1 Patient and transplant characteristics in autologous recipients for sub-analysis………...54
x LIST OF ABBREVIATIONS
ANOVA Analysis of variance
AP Acute Pancreatitis
AP-1 activator protein 1
APC Antigen Presenting Cells
ASC Apoptosis associated Speck-like protein
ATF Activating transcription factor
BD Brain dead
BSA Bovine serum albumin
CAPS Cryopyrin associated periodic syndromes
CARD Caspase activation and recruit domains
Casp Caspases
CAY CAY 10512 (potent analog of reservatrol)
CCL CC chemokine ligand
CD cluster of differentiation cDNA complementary deoxyribonucleic acid
CER Cerulein
CFTR Cystic Fibrosis transmembrane conductance regulator
CHOP C/EBP homologous protein
CIT Consortium of islet transplantation
xi CMRL Connaught Medical Research Laboratories
CO2 Carbon dioxide
CP Chronic pancreatitis
C-peptide connecting peptide
CXCL Chemokine (C-X-C motif) ligand
CXCR Chemokine(C-X-C motif) receptor
CYT Cytokine cocktail
DAB 3,3'-Diaminobenzidine
DAMPs Damage associated molecular patterns
DAPI 4',6-diamidino-2-phenylindole
DC Dendritic cell
dsDNA double stranded DNA
Egr Early growth response protein
Eif2a Eukaryotic translation initiation factor 2a
ELISA enzyme linked immunosorbent assay
EP3 Prostaglandin E receptor 3
ER endoplasmic reticulum
ERK Extracellular signal-regulated kinases
FIH Factor inhibiting HIF
FITC Fluorescein isothiocyanate
G-CSF Granulocyte-colony stimulating factor
H&E Hematoxylin and Eosin
HbA1c Hemoglobin A1c
xii
HFD High Fat diet
HIF Hypoxia inducible factors
HLA human leukocyte antigen
HMGB1 High mobility group box-1
HO-1 Heme oxygenase 1
hrs hours
HSP Heat shock proteins
i.p. intraperitoneal
IACUC Institutional Animal Care and Use Committee
IBMIR Instant Blood Mediated Inflammatory Reaction
IEQ islet equivalents
IFN Interferon
IgG Immunoglobulin G
IgM Immunoglobulin M
IHC immunohistochemistry
IKKβ IκB kinase β
IL Interleukin iNOS inducible Nitric oxide synthases
IP-10 Interferon gamma inducible protein 10
IκB inhibitor of Kappa B
JNK c-Jun N-terminal kinases
LMWDS low molecular weight dextran sulphate
LPS lipopolysaccharide
xiii MAPK mitogen-activated protein kinase
MCP1 monocyte chemotactic protein 1
MIF Macrophage migration inhibitory factor mins minutes
MIP1α Macrophage inflammatory protein 1α miR miRNA miRNA microRNA
MKK MAPK kinase mmHg millimeter mercury mRNA messenger RNA
NALP3 NACHT, LRR and PYD domains-containing protein 3
NEMO NFκB essential modulator
NFAT Nuclear Factor of activated T cells
NFκB Nuclear Factor Kappa B
NKT natural killer T cells
NLR NOD-like receptors
NLRP3 NOD-like receptor family, pyrin domain containing 3
NOD non-obese diabetic
PAMPs Pathogen associated molecular patterns
PCR polymerase chain reaction
P-ERK phosphorylated ERK
PERK protein kinase R (PKR)-like endoplasmic reticulum kinase
PHD Prolyl hydroxylase
xiv
PMN Polymorphonuclear cells
pO2 partial pressure of oxygen
PP Pancreatic polypeptide
qRT quantitative realtime
RNA ribonucleic acid
rRNA ribosomal RNA
SPINK1 serine protease inhibitor Kazal-type 1
T1D Type 1 diabetes
TAT Thrombin-antithrombin
TBS Tris Buffered Saline
T-ERK total ERK
TF Tissue Factor
TLR toll like receptors
TP total pancreatectomy alone
TPIAT total pancreatectomy auto islet transplantation
TRITC Tetramethylrhodamine
TUNEL Terminal deoxynucleotidyl transferase dUTP nick end labeling
UPR Unfolded protein response
WA Withaferin A
XBP1 X-box binding protein 1
xv ACKNOWLEDGMENTS
First and foremost, I would like to thank Almighty GOD for giving me the strength and determination to complete my dissertation successfully. I would like to thank my family members for their considerable support and encouragement, without which this may not have been possible.
I am truly grateful to my mentor Dr. Bashoo Naziruddin for believing in me and guiding
me through the course of my research. I have learned a lot under his guidance and I wholeheartedly
appreciate his unceasing motivation that kept me moving forward. He has not only been a mentor
to me but also a great friend to share my ups and downs in research. He has always been optimistic
and taught me the same approach.
I would like to thank Baylor University and the graduate program for giving me the
opportunity to become a doctoral candidate and providing me with the support for doing research.
I am grateful to Dr. Robert Kane and the department of biomedical studies for making me a part
of their graduate team. I am sincerely thankful Dr. Kane for being there to answer any of my
questions both scientific and department related.
I would like to extend my humble gratitude to my committee members Drs. Christopher
Kearney, Sangkon Oh, and Michael C. Lawrence for being there to strengthen my dissertation to
the fullest. I would especially like to acknowledge Dr. Lawrence for his time and ideas that helped
me develop my research.
I have been blessed with the best lab members and colleagues who have supported me, and
helped me in every step of my research. There are so many people I would like to acknowledge
my gratitude but I may not be able to thank everyone specifically. Although, I would like to
xvi acknowledge some key members who have helped me get through this difficult path very easily. I would like to thank Drs. Levy, Itoh, Shahbazov, Faisal, Gumpei and many others. I would also like to acknowledge my colleagues Jeff Sorelle, Omar Khan, Ana Rahman, Yoshiko Tamura, Nofit
Borenstein, Regina Whitaker and last but not the least Dr. Carson for helping me with the completion of my course requirements for PhD. Finally, I would like to thank each and every member who has been a part of life and has helped me through my career path.
xvii DEDICATION
To my loving family
xviii CHAPTER ONE
Introduction
Part of this chapter published as: Kanak MA, Takita M, Kunnathodi F, Lawrence MC, Levy MF, Naziruddin B. Inflammatory response in islet transplantation. Int J Endocrinol 2014;2014:451035.
The pancreas is a vital organ of the gastrointestinal tract responsible for exocrine and endocrine secretions required to digest food and maintain glucose homeostasis.
Approximately 97% to 99% of the adult pancreas is the exocrine compartment (acinar cells), and about 1% to 3% is the source of endocrine cells (islets of Langerhans)1. The acinar cells produce a number of enzymes that are required for the digestion of food. The role of islets of Langerhans is quite different from that of acinar cells; their major function is to maintain glucose homeostasis. Islets of Langerhans are a cluster made up of at least 4 different types of endocrine cells. Most cells in the islet are β cells, which are responsible for production of insulin. The next most abundant cell type is α cells, which produce glucagon, which is also required for the maintenance of glucose homeostasis.
Other cell types that are present in an islet are δ cells and PP cells, which synthesize somatostatin and pancreatic polypeptide, which are required for absorption of food and appetite, respectively2. Both the exocrine and the endocrine compartments are prone to inflammation, which could result in diseases such as pancreatitis and/or diabetes.
This chapter reviews islet transplantation as treatment for two diseases of the pancreas: type 1 diabetes (TID) and chronic pancreatitis (CP). It highlights the inflammatory response in islet transplantation in preparation for Chapter Two, which
1 presents a study of alleviation of the instant blood-mediated inflammatory reaction
(IBMIR) through treatment of islets with nuclear factor-kappa B (NFκB) inhibitor.
Chapter Three continues the focus on clinical islet transplantation, evaluating
microRNA375 as a novel biomarker for graft damage. Chapter Four moves to the disease
of CP and presents research on use of NFκB inhibitor to prevent disease progression. The
dissertation closes with a summary and conclusions.
Diseases of the Pancreas
Type 1 Diabetes
T1D is an autoimmune disease that leads to destruction of the β cells of the
pancreas3. Due to the lack of β cells, patients with T1D suffer from hyperglycemia and other long-term complications related to it. T1D has afflicted more than 135 million people worldwide and in the United States, and an incidence of 15 to 18 per 100,000 people has been recorded per year4. The major focus of research in this field is on understanding triggers that lead to the onset of T1D, which is critical to strategize treatment options. Currently, exogenous administration of insulin is the prescribed therapy to maintain normoglycemia5. T1D has been known to cause chronic
complications, which are linked to morbidity and mortality6.
A major symptom of the disease is the unpredictable changes in glycemic levels, with sudden hyper- and hypoglycemic conditions that may result in unconsciousness, coma, and even death7. This condition, called “brittle” diabetes, can only be treated by
replacing the patient’s β cells with functioning β cells by whole-organ pancreas
transplantation or islet cell transplantation8. Although pancreas transplantation is a
2 promising therapeutic option, the surgery is complicated with an increased risk of
mortality9. Islet cell transplantation is growing in recognition as a potential treatment option for T1D patients because it restores functional β-cell mass, reduces the risk of long-term complications of diabetes, abolishes hypoglycemic episodes, and most importantly is a minimally invasive procedure10.
Chronic Pancreatitis
CP is a progressive inflammatory condition that leads to pancreatic exocrine insufficiency and irreversible damage of the pancreatic parenchyma11. In its early stages,
the disease affects pancreatic exocrine function, which could be followed by impaired
endocrine function that results in the onset of diabetes mellitus, also referred to as type 3c
diabetes12, 13. The mechanisms underlying the development of CP are not clearly defined.
Inflammation, heavy alcohol consumption, pancreatic ductal obstructions, calcification,
sphincter of Oddi dysfunction, certain genetic mutations, and periampullary tumors are
considered the major causes of CP. In addition to gradual loss of exocrine and endocrine
function, complications of CP include biliary or duodenal stenosis and intractable pain.
Total or partial pancreatectomy followed by intrahepatic transplantation of
autologous islets has emerged as a promising approach to treat CP, due to its ability to
reduce or eliminate pain while retaining endocrine function. The first pancreatectomy
followed by autologous islet transplantation was performed at the University of
Minnesota in 197714. Since then, more than 500 CP patients have successfully undergone
this procedure at several centers in North America and worldwide15. At 3 years
posttransplant, 30% of patients were insulin independent, 33% had partial islet function,
and 94% showed improved pain control16.
3 Islet Transplantation
Transplantation of pancreatic islets, a minimally invasive procedure involving
infusion of islet cells into the portal vein of the liver, was first demonstrated in an
experimental diabetic model by Lacy et al. in 197317. Results from the initial clinical
trials for T1D were only partially successful, with only 10% of the patients achieving
insulin independence at 1 year posttransplantation18. A quantum leap in islet transplantation occurred when the Edmonton group introduced a steroid-free immunosuppression and showed insulin independence in all 7 of their patients19. With
this advancement in immunosuppression and the continuous improvement in islet
isolation techniques, islet transplantation has entered a new era of heightened success.
Currently, two types of clinical islet transplantation are performed: allogenic and
autologous. Allogenic islet transplantation is typically performed on patients with severe
T1D, while autologous islet transplantation is performed on patients suffering from
severe CP and undergoing partial or total pancreatectomy. Compared with similar doses
of allogeneic islets, survival of autologous islets is far better over time16. In contrast to
allogeneic islet transplants, autologous islet transplants are devoid of an autoimmune
response and alloimmune rejection, and recipients do not require β-cell–toxic immuno- suppressive drugs. Similar to intrahepatic allogeneic islet transplants, however, autologous islet transplants are subject to ischemic, hypoxic, and innate inflammatory damage.
Causes of the Inflammatory Response in Islet Transplantation
Despite recent progress in clinical outcomes, several obstacles for allogenic and
autologous islet transplantation still need to be addressed. These include improvement in
the technical aspects of the isolation procedure; improvement in the quality of isolated
4 islets; reduction of inflammation during the peritransplant period triggered by incompatibility between islets and the blood interface, known as IBMIR; and improvement in the long-term survival of transplanted islets. One common underlying cause in each of the obstacles to clinical success is the production and secretion of inflammatory mediators, which induce apoptosis and impair function of transplanted islets. This section addresses the causes of inflammation in islets, including inflammation related to donor factors, islet isolation, pretransplant islet culture, the transplant itself, and posttransplant factors (Figure 1.1). Special attention is given to IBMIR and the center’s study of IBMIR in autologous islet transplantation. The sections that follow highlight strategies to reduce inflammation and discuss future directions.
Donor-induced inflammation Post-transplant hypoxia Cytokine storm Ischemia injury
Islet isolation Complement activation Pancreas digestion Hypoxia Mechanical stress Coagulation
Peri-transplant inflammation Activated innate immunity Cytokine induced
Figure 1.1. Factors that induce an inflammatory reaction to islet grafts.
5 Pancreas Donors
Damage to the islet cells begins with the donor. Immune cell infiltrates and
inflammatory mediators are produced in the donor pancreas upon brain death. Most islet
transplants use organs from heart-beating, brain-dead cadaveric donors. Organs from
brain-dead donors have much better outcomes than organs from non–heart-beating
donors. However, acute physiological changes after brain death in brain-dead donors may
still result in significant damage to the islets from inflammatory events. Indeed, several
reports have documented poor transplantation outcomes due to organ damage caused by
extremely high levels of inflammatory cytokines in the circulation20-23. This systemic
elevation of inflammatory cytokines, the so-called “cytokine storm” in brain-dead donors,
is due in part to hemodynamic and metabolic changes occurring after brain death24.
Inflammatory cytokines are effective mediators of β-cell dysfunction and death25.
Recent studies have indicated methods to improve islet graft function by
inhibiting inflammatory mediators in brain-dead donors upon procurement. One study
showed that maintaining normoglycemia after brain death by insulin reduces
inflammatory shock26. Transfusing sivelestat sodium, a selective neutrophil elastase
inhibitor, was shown to increase islet yield, protect islet function and quality, and inhibit
hypercytokinemia-mediated β-cell death in a brain-death rat model27.
Ischemic Time
Islets are subjected to hypoxic conditions from the onset of ischemia until graft revascularization. Hence, ischemic time is a major concern that needs to be addressed to
improve islet quality for transplantation. Since islets are physiologically adapted to
receiving an ample supply of oxygen, prolonged ischemic times increase damage to
6 islets. A previous study showed that isolations performed with a pancreas with a cold ischemic time of >8 hours produced significantly lower islet yields, with impaired islet function28. The quality of the islets can be improved by reducing organ ischemic time.
Another strategy used to reduce islet ischemic damage has been to maintain the procured pancreas in a high-oxygen-content solution. A mixture of University of Wisconsin solution along with perfluorocarbon, which has high affinity for oxygen, has been used to reduce islet damage by hypoxia29.
The viability of islets is significantly reduced due to depletion of ATP and apoptosis induced by hypoxia30. Hypoxia in islets activates NFκB signaling, induces transcription of inducible nitric oxide synthase (iNOS), and increases expression of monocyte chemotactic protein 1 (MCP-1), leading to infiltration of macrophages and destruction of islets31. During hypoxia, islets try to compensate for the low oxygen availability by activating the transcription factor hypoxia-induced factor (HIF)32. When activated, the HIF-1α and HIF-1β subunits translocate to the nucleus and bind to the hypoxia response element. This induces transcription of several genes, including toll-like receptors. Under normal conditions, HIF activity is rendered inactive by prolyl hydroxylases and factor-inhibiting HIF, both of which prevent the activation of inhibitor of kappa B (IκB) kinase B and hence inhibit NFκB activation33-37.
Islet Isolation Process
Isolation of high-quality islets in sufficient quantity remains a major impediment for the success of this procedure, due to chronic inflammatory conditions affecting the pancreas. The enzymes used for islet isolation could be a factor in the lack of satisfactory results in islet transplantation. It is well documented that enzymatic and mechanical stress
7 can induce inflammatory mediators in islets. Gene array studies have shown upregulation of genes related to inflammation, apoptosis, cell growth, and angiogenesis immediately after isolation, with a further increase after 72-hour culture. The most highly upregulated genes have been the ones for inflammation and apoptosis. Isolation and culture of islets have also been shown to downregulate expression of interleukin (IL)-10, which is a negative regulator of cytokine expression and also induces development of regulatory T cells. Conversely, isolation and culture of islets have upregulated the expression of IL-8, which is a potent inflammatory and angiogenic factor, and the expression increased several fold after 3 days in culture38. IL-8 has been shown to play a negative role in the longevity of the graft in kidney, liver, and lung transplants; overexpression of IL-8 has been associated with poor graft life39-41. Gene array analysis of cultured islets has also shown that the top 15 upregulated genes were all induced by the NFκB pathway38.
Therefore, inhibiting the NFκB pathway during isolation and/or culture might improve islet quality for transplantation.
Pretransplant Islet Culture
There has been a continuous debate as to which islets are superior: freshly isolated or cultured islets. Several reports have shown that freshly isolated islets are better than islets cultured for at least 24 hours42-44. A recent report from our center confirmed that freshly isolated islets outperformed cultured islets to address concerns over the rapid deterioration of islet counts after culture. The study showed a decrease in the islet counts by 13%, 24%, and 35% on days 1, 2, and 3, respectively. When transplanted into diabetic nude mice, the achievement of normoglycemia was significantly improved with fresh islets compared to cultured islets45, 46. It has been reported that culturing islets can lead to
8 overexpression of inflammatory mediators caused directly by islets, by stimuli in the
culture medium, or by contamination of exocrine tissue in the culture47. During culture,
islets have to depend on oxygen through diffusion, but the core of the islet begins to
undergo hypoxia, causing upregulation of genes induced by oxidative stress and
apoptosis48. Islets in the culture have been shown to induce expression of proteins such as
tissue factor and MCP-1; this induction could be abrogated by the use of nicotinamide in
the culture media49. Still, there remains the argument that culturing islets before transplantation will improve the purity of the islets, as acinar cells and damaged islets would die in the culture. It has also been suggested that providing a culturing period would allow islets to recover from the stress of the isolation process, although there is no
compelling data to support this hypothesis. Nonetheless, the Consortium of Islet
Transplantation has adopted pretransplant culture as a standard procedure for clinical
transplantation50.
Peritransplant Inflammation: IBMIR
One of the major causes of islet destruction during transplantation is IBMIR
(Figure 1.6). Bennet et al. first proposed that when freshly isolated “naked” islets come into direct contact with the ABO-compatible allogenic blood, activation of an innate
immune reaction ensues51. Tissue factor expressed on islets could be a major trigger for
such reactions. The islets, along with resident antigen-presenting cells (APCs), secrete cytokines and chemokines, which play a major role in the inflammatory process.
Infiltration of leukocytes and macrophages initiates destruction of the islet cells before engraftment51, 52. Blocking tissue factor using antibodies significantly reduced the clotting
reaction52.
9 IBMIR and Autologous Islet Transplantation
Although previous studies showed induction of IBMIR under allogenic and
xenogeneic conditions51, 52, our group showed for the first time that IBMIR was also an
issue during autologous islet transplantation. Blood collected from clinical transplant
patients at the time of transplantation was used to analyze markers of coagulation,
complement activation, and inflammatory cytokine secretion53. To demonstrate the
mechanism of IBMIR in vitro, I developed a miniaturized tube model. The model was
modified from the in vitro blood loop model developed by Korsgren et al54. Islets come in direct contact with the blood of the portal vein and flow with the bloodstream until they lodge in the liver. To mimic the same scenario of islets in the intraportal milieu, isolated human islets were added to autologous human blood in a heparinized Eppendorf tube and then the cells were placed in a 37°C shaker incubator and the plasma samples were collected at various time points (Figure 1.2). The in vitro miniaturized tube model was validated by measuring parameters such as partial pressure of oxygen, pH, and the glucose content of blood for up to 6 hours in the presence or absence of islets. The data suggested that the in vitro model was suitable for investigating IBMIR for up to 6 hours
(Figure 1.3). Results obtained from the in vitro experiment corroborated results of blood
samples from clinical autologous islet transplant patients, suggesting that IBMIR
occurred in autologous islet transplantation (Figures 1.3 and 1.4).
10 Figure 1.2. Flow diagram of the method for in vitro IBMIR using a miniaturized tube model.
Figure 1.3. Data demonstrating autologous IBMIR activation in vitro and validation parameters. The mixed blood and islet group (solid line) had significantly increased TAT (A) and C-peptide (B) levels (*p with repeated measured ANOVA < 0.05) although the blood-only (dotted line with squares) and islet-only samples (dotted line with triangles) did not. C3a levels were elevated in both the mixed blood-islet and the blood-only samples, but not in the islet-only samples (C). Culture conditions on pH (B), pO2 (D) and glucose level (F) were determined. The mixed blood and islet group showed similar trend of pH and glucose level to blood only group. Although pO2 levels were significantly lower in mixed group than blood only group (*p<0.05, D), pO2 level over of 60 mmHg was maintained for 6 hours in the mixed group. Data are expressed as mean ± SE.
11 Figure 1.4. Elevation of proinflammatory cytokines and chemokines in serum after IBMIR. The mixed blood and islet group had significantly higher proinflammatory cytokine levels of MCP-1 (A) and TNF-α (B) in culture for 6 hours when compared to blood only group (*p with repeated measurement ANOVA <0.05). Data are expressed as mean ±SE.
Serum samples collected from patients undergoing total pancreatectomy with islet
autotransplantation were collected at various time points during and after islet infusion.
Thrombin-antithrombin levels increased in these samples immediately after infusion of
islets, suggesting activation of the coagulation pathway; a decrease in platelet number
also suggested the same. C-peptide has been regarded as a marker for islet damage during
infusion. Elevation of C-peptide was seen immediately after infusion, suggesting that
islets were being destroyed in the mechanism. Interestingly, in contrast to allogeneic and
xenogeneic transplants, autologous islet transplantation did not cause complement
activation (Figure 1.5). This may suggest that the mechanism of IBMIR may be less
severe in the case of autologous islet transplantation; nonetheless, its effects are drastic.
Mechanisms Underlying IBMIR and Strategies to Block IBMIR
The IBMIR effect has been characterized by an immediate increase in the
thrombin-antithrombin levels after islet infusion and in the levels of C-peptide in the
12 blood. A recent clinical study utilized labeled islets combined with positron emission tomography and computed tomography and demonstrated that about 25% of the islets are lost immediately after transplantation55. IBMIR is also characterized by coagulation, complement activation, secretion of chemokines that attract innate immune cells, and release of proinflammatory cytokines leading to islet damage by apoptosis56. The mechanisms underlying IBMIR and methodologies to block this phenomenon are currently being investigated, as described below.
Figure 1.5. Activation of IBMIR in samples from patients undergoing total pancreatectomy with islet autotransplantation. Significant differences in TAT levels were observed for the middle of infusion to 1 hour after infusion when compared to the pre-infusion level (*p with repeated ANOVA < 0.05) (A). C- peptide levels for middle of infusion to day 7 were significantly higher than prior to infusion (*p <0.05, C); however, no significant differences were found in C3a and C4d levels (B and D). sC5b-9 levels had two peaks at islet infusion and day 5; however, these changes were within normal range and no significant differences were observed during peritransplant period up to day 7 when compared to pre-infusion level (F). Platelet counts significantly decreased at 1 hour to 3 days after infusion (*p <0.05) (E). Shadow area shows normal range. Data are expressed as mean ± SE for 10 patients.
13 Figure 1.6. Hypothetical model showing the mechanism of instant blood-mediated inflammatory reaction. IgM indicates immunoglobulin M; MCP-1, monocyte chemotactic protein 1; PMN, polymorphonuclear leukocytes; TF, tissue factor.
Coagulation: Coagulation is activated as soon as the islets come in direct contact with the portal blood during transplantation. The coagulation occurs through the extrinsic pathway, which requires tissue factor for activation. Tissue factor expressed on the surface of isolated islets interacts with factor VIIa and initiates the coagulation cascade by formation of thrombin and generation of fibrin clots52, 57, 58. The production of thrombin at high concentrations can exacerbate the destruction process because at high concentrations thrombin is proinflammatory and an inducer of apoptosis59, 60.
14 Nilsson et al. showed that the use of anticoagulants such as melagatran could
significantly reduce IBMIR in vitro in a blood loop model54. Johansson et al. previously
showed that low-molecular-weight dextran sulfate (LMWDS), a potent inhibitor of
coagulation and complement activation, could efficiently suppress IBMIR. They used an
in vitro tubing loop model to investigate the effect of LMWDS on islets when exposed to
human blood, showing effects on activated partial thromboplastin time, platelet function,
and complement activation61. Other strategies to block IBMIR that have been intensely
investigated have included encapsulating islets or coating the islet surface with inhibitory
molecules to promote successful islet engraftment. The islet surface has been coated with
heparin complex by exploiting biotin-avidin chemistry to inhibit coagulation62.
Endothelial cells have also been used to coat the islets; transplantation of these coated
islets has reduced coagulation, complement activation, and leukocyte infiltration63, 64.
Alternatively, the use of mesenchymal stem cell–coated islets has been shown to promote endothelial cell proliferation, revascularization, islet neogenesis, and immune modulation65. Another study showed that IBMIR could be targeted by overexpressing
CD39, which is an ectonucleotidase that degrades ATP required for platelet activation in the islets. In vivo and in vitro experiments showed less coagulation activity after transplantation using islets from CD39 overexpressing transgenic mice66.
Complement activation: Another major component of IBMIR is the activation of
the complement cascade. Complement activation is shown by the increase in the
concentration of complement proteins in the serum of islet transplant recipients56, 67.
Activation of complement protein C3a and C5a leads to recruitment and accumulation of
leukocytes, upregulation of adhesion molecules on the endothelium and platelets, and
15 production of reactive oxygen species (ROS) and cytokines67. Also, the accumulation of
IgG, IgM, and complement proteins C3, C4, and C9 on the surface of human islets
immediately after treatment with ABO-compatible blood indicates that immunoglobulin
is involved in the activation of the classical complement pathway67.
Complement protein C5a has been proposed as a critical molecule responsible for
activation of coagulation and inflammation by mediating tissue factor expression in
neutrophils68, 69. A recent short report from Tokodai et al. showed that the combined
effect of anticoagulant gabexate mesilate and complement protein C5a inhibitory peptide
successfully improved islet transplantation compared to individual use of the drugs in a
syngeneic rat transplant model70. Thrombin-antithrombin levels were also reduced with
the combined treatment. Tissue factor was downregulated by complement protein C5a
inhibitory peptide treatment, but only on the liver granulocytes and not on the islet
grafts71.
The use of a complement inhibitor such as compstatin has been shown to
significantly improve graft survival in in vivo models72. In a recent study, it has been
reported that immobilization of soluble complement receptor on the islet surface using
polyethylene glycol inhibited complement activation significantly and protected islets
from damage due to xenoreactive antibodies73. In clinical autologous islet transplantation,
complement proteins have no influence during IBMIR53.
Cytokine secretion, leukocyte infiltration, and β-cell damage: Syngeneic transplant models have shown that the damage to islet grafts is primarily due to a nonspecific inflammatory response74, 75. Similarly, clinical data on autologous islet
transplantation on patients with CP showed elevated levels of proinflammatory cytokines
16 immediately posttransplantation53. The islet resident macrophages, Kupffer cells, and
neutrophils secrete IL-1β, which impairs insulin secretion and induces islet cell
apoptosis76-78. IL-1β activates NFκB signaling and induces expression of several other
inflammatory mediators, including IL-1β, IL-8, IP-10, IL-6, tumor necrosis factor (TNF)-
α, MCP-1, intercellular adhesion molecule-1, vascular cell adhesion molecule-1, endothelial leukocyte adhesion molecule-1, iNOS, prostaglandin E2, and prostaglandin
EP379-82. These mediators further induce a cascade of inflammatory events that have deleterious effects on islet graft survival.
In addition to several proinflammatory cytokines, islets are known to express chemokines after isolation and during culture, of which CXCL10 (IP-10), CXCL8 (IL-8),
and CCL2 (MCP-1) are the major ones38, 56. IP-10 is widely recognized as a chemokine
that attracts leukocytes to the site of infection or inflammation. On the other hand, IL-8
and MCP-1 are known to recruit neutrophils and macrophages, respectively. The role of
chemokine receptors and ligands in various transplant rejections has been reviewed
elsewhere83.
In the context of islet transplantation, it has been shown that allograft function is
prolonged under anti-CXCL10 antibody therapy due to the lack of infiltration of
leukocytes to the graft site84. Using CCL2–/– knockout mice, it was demonstrated that
reducing CCL2 expression in recipients is more important than knocking out CCL2 in
donors. Knocking out CCL2 in the recipient also resulted in abrogation of other
chemokines (CXCL1, CCL4, CCL6, and CXCL9) that were upregulated in the liver
posttransplantation85. A recent approach using reparixin, an inhibitor of CXCR1/2, has
shown an improved islet transplant outcome in allogeneic mouse models. Furthermore, a
17 pilot study on human islet allotransplant patients also showed promising results.
Comparison of reparixin-treated group with nontreated controls showed a reduced insulin requirement, lower hemoglobin A1c levels after transplantation, and improved fasting C- peptide levels. The proposed mechanism is the lack of recruitment of polymorphonuclear leukocytes and natural killer T cells to the liver86. Based on these reports, it is conceivable that reducing secretion of proinflammatory cytokines and chemokines during the initial stages of transplantation could improve islet transplant outcomes.
Several reports have shown that allogeneic islet transplant recipients develop antibodies to both human leukocyte antigen (HLA) class I and II antigens of donor origin87-89. While it is evident that islets constitutively express HLA class I antigens, the expression of HLA class II on isolated human islets has not been clearly demonstrated.
Upon stimulation with inflammatory cytokines of TNFα and interferon (IFN)-γ, islet- enriched pancreatic cultures and MIN6 cell lines have been shown to express HLA class
II antigens90, 91.
Recently, our group has demonstrated that under inflammatory conditions, there is induction of HLA class II expression on the islet cell surface leading to the development of anti-donor class II antibodies in the recipient92. Islets isolated from deceased donor pancreases were used to demonstrate induction of HLA class II molecules through real- time polymerase chain reaction analysis, immunofluorescent staining, and flow cytometry. One of the transplant recipients developed antibodies to a mismatched donor
HLA class II allele (DR7). Posttransplant serum from this patient showed significant binding to cytokine-stimulated islet cells from DR7-positive donors only. These results clearly demonstrate that human islets express HLA class II under inflammatory
18
conditions. This could lead to an anti-class II response, which in turn may play a critical
role in rejection of islet allografts.
Posttransplant Inflammation
Hypoxia
Like other transplantable organs, islets are prone to ischemic reperfusion injury.
When isolated islets are infused into the blood circulation, several cytotoxic products are
produced due to oxygen metabolism, which is catalyzed by xanthine oxidase93.
Activation of this enzyme results in the formation of superoxide and hydrogen peroxide,
which can interact with free iron and copper radicals to produce cytotoxic ROS94. ROS can induce cell death by damaging and degrading proteins, lipids, and nucleic acids and activating NFκB inflammatory pathways95. Βeta cells of the islets are more vulnerable to
attack to ROS because of their low expression of antioxidants, including glutathione
peroxidase, catalase, and superoxide dismutase96-98. Indeed, increasing the expression of
cellular antioxidants and treatment with exogenous antioxidants have both been shown to
protect islets in a diabetes models99, 100.
Following intraportal infusion, there is a low oxygen supply at the graft site in the liver, creating a hypoxic environment for the islets101, 102. Islet cells have counter
mechanisms to restore the oxygen supply32. Eventually, islet grafts begin to vascularize, but the amount of oxygen supply even after hepatic vascularization is significantly lower than in the pancreas (5 mm Hg as opposed to 40 mm Hg)103. Although the liver is
currently the preferential site for islet transplantation, it is still not considered the optimal
host site because of the lack of oxygen; therefore, other sites that might improve oxygen
19 availability to the islets are still under consideration. Cantarelli et al., for example, have provided evidence that bone marrow may be a more suitable graft site for islet transplantation because of the increased vasculature and abundant supply of oxygen104.
Furthermore, pilot studies on clinical autologous islet transplants have been performed in bone marrow which outperformed islet grafts in the liver105.
Innate Immunity
Pancreatic islet inflammation after transplantation is induced by innate immune cells such as neutrophils, islet resident macrophages, and Kupffer cells. Kupffer cells can induce damage to other cells by releasing free radicals and secreting proinflammatory cytokines106. The amount of proinflammatory cytokines IL-1β and TNFα secreted by
Kupffer cells is increased after islet transplantation107. This inflammatory cytokine elevation is blocked in the case of a macrophage-depleted recipient. Several factors have been postulated to explain the activation of Kupffer cells in the liver after islet transplantation. One report suggested that the collagenase used for islet isolation contains endotoxin, which could activate production of proinflammatory cytokines by islet- resident macrophages, and in turn activate Kupffer cells108. The presence of acinar tissue in the islet transplant preparation has also been investigated as the cause for activation of
Kupffer cells. It has been demonstrated that the amount of exocrine contamination is directly correlated to posttransplant necrosis and lower viability of pancreatic islet cells.
It has been postulated that when these acinar cells die, they release enzymes that trigger an inflammatory response leading to islet cell death109.
Another hypothesis is that the professional APCs residing inside the islets are responsible for inducing the inflammatory response. A recent study on the intraislet
20 immune cell population showed that 67% of the immune cells in the islets are APCs, and
50% of these APCs are B cells110. Use of anti-inflammatory agents that block the
production and secretion of these proinflammatory cytokines and chemokines would be a
useful strategy to protect islets from innate immune cell-mediated damage. Recently we
have shown that use of withaferin A (WA), which is an NFκB inhibitor, protected islet
viability, reduced secretion of proinflammatory chemokines and cytokines, and reduced
infiltration of neutrophils in an in vitro tube model111.
Inflammatory Response from Islets
Islets can also produce inflammatory mediators in response to metabolic, inflammatory, physical, and chemical stress. For example, IL-1β induces production of
cytokines, chemokines, and iNOS in pancreatic β cells112-114. In the context of islet
transplantation, multiple islet stress events may contribute to cascading cytokine-induced
cytokine production in islets. Thus, cytokines produced and released by β cells
throughout the islet transplant process may exacerbate islet inflammation.
MAP kinases ERK1/2, p38, and JNK have roles in islet dysfunction and apoptosis
in response to stress-specific signaling pathways115, 116. Calcineurin-dependent ERK1/2
activation has short-term stimulatory effects on insulin production and islet function in
response to glucose and secretagogues; however, sustained activation due to metabolic
and inflammatory stress may have deleterious effects117-120. Upon exposure to cytokines,
JNK and p38 are activated by MAP kinase kinase (MKK) 4 and MKK3/6,
respectively116. Additionally, exposure of islets to stress from the islet isolation process
activates JNK and p38 via MKK7 signaling. One study showed that an increased
p38/JNK activation relative to ERK1/2 activation after islet isolation correlates with
21 decreased islet survival121. Hence, identifying methods to selectively block p38 and JNK
pathways, while preserving acute ERK1/2 signaling, may enhance islet graft function and
survival in islet transplantation122-126.
Acute exposure of islets to a low dose of IL-1β has been shown to stimulate
proliferation and enhance β-cell function, whereas exposure to high-dose IL-1β impairs
secretion and induces apoptosis127-129. Induction of TNFα expression in β cells by IL-1β
requires activation of calcineurin and downstream transcription factor nuclear factor of
activated T cells (NFAT)112. Extended exposure of β cells to IL-1β results in sustained
MAP kinase signaling, which activates NFκB-dependent genes that are proapoptotic112,
130, 131. Although a number of reports suggest that NFκB is responsible for apoptosis of transplanted islet cells132, other reports have presented conflicting evidence suggesting
that NFκB prevents islet cell death133. This is also likely attributed to the strength and
duration of exposure as well as the type of stimulus, as both NFAT and NFκB have
overlapping roles in cytokine gene expression and β-cell proliferation and function112, 133-
136. Thus, minimizing sustained activation of MAP kinase and NFAT/
NFκB signaling during the transplantation process is likely key to reducing islet
inflammation and improving islet transplant outcomes.
Antiinflammatory Strategies to Improve Islet Transplantation
Antiinflammatory agents have been incorporated into immunosuppressive
regimens in recent clinical allogeneic islet transplant protocols137, 138. A recent review has
summarized progress related to this approach24. In the era of 1999 to 2002, TNFα
inhibitors were utilized in a small proportion (11.8%) of islet transplants, while in the
2007 to 2010 period, they were used in 33.8% of transplants139. The first report of islet
22
transplantation involving an antiinflammatory agent came from the University of
Minnesota, where etanercept was incorporated in peritransplant immunosuppression
therapy140. All the islet recipients achieved insulin independence after a single islet
infusion. The University of Miami group implemented an immunosuppression protocol
containing infliximab, which resulted in a high success rate of insulin independence
(87.5%)141. Recently, a meta-analysis showed significant improvement in the insulin
independence rate when a TNFα inhibitor was coupled with a T-cell depletion regimen,
compared with T-cell depletion only142. The success rate with T-cell depletion
immunosuppression plus a TNFα inhibitor is now likely comparable to that of whole-
organ pancreas transplantation alone, offering approximately 50% success at 5 years
posttransplant139, 142, 143.
No significant difference in the success rate, however, was seen between a T-cell–
depleting islet protocol with and without infliximab in a clinical study141. In an
immunodeficient rodent model transplanted with a marginal mass of human islets, no
significant benefits in islet engraftment were observed when only etanercept was given,
whereas significant improvement in diabetes reversal was demonstrated when both
etanercept and anakinra, an IL-1β receptor antagonist, were administered144. Thus,
administration of a TNFα inhibitor alone might be insufficient to fully control
peritransplant inflammation. Interestingly, our group has implemented combined therapy
of etanercept and anakinra in clinical allogenic islet transplantation and has shown that all
patients achieved insulin independence after a single infusion, although the number of
islet recipients was limited145. A large, well-designed clinical study is required to evaluate the impact of these antiinflammatory agents in islet engraftment. The use of
23 antiinflammatory agents in clinical allogeneic islet cell transplantation is summarized in
Table 1.1.
A major requirement in clinical islet transplantation is to identify a biomarker that can be used to evaluate islet damage during and after transplantation. Currently, the tools that are used as islet damage markers are serum levels of C-peptide and proinsulin that can easily be altered depending on the metabolic state of the patient. C-peptide and proinsulin are both functional proteins that can be released upon glucose stimulation, and these can also yield false positive results if used as damage markers for islet transplantation.
Several antiinflammatory compounds are currently being investigated and more are emerging to improve the outcomes of islet transplantation. An exhaustive review on the strategies and approaches to improve clinical islet transplantation has been mentioned24. Pileggi et al. have shown that expression of heme oxygenase-1 (HO-1) in islet cells protected the islets from apoptotic death. To induce HO-1 in islets, the cells were cultured in ferriprotoporphyrin IX chloride and cobaltic protoporphyrin IX chloride before transplantation146. The HO-1 gene plays an important role in iron homeostasis, but also behaves as a potent antiinflammatory and antiapoptotic gene147, 148. A similar study has shown the antiapoptotic effect when islets are treated with carbon monoxide. This treatment causes islets to produce cytoprotective cyclic guanosine monophosphate and its downstream kinases149. Another strategy that has shown promise is the expression of the
A20 gene in islets by ex vivo gene transfer, which protects these islets from cytokine- induced damage. A20 is an inhibitor of NFκB, which is a transcriptional regulator of several proinflammatory cytokines150. A different study involving the use of diannexin,
24 which is a homodimer of annexin 5, has demonstrated a significant decrease in β-cell apoptosis and improved graft function after in vivo transplantation151. The use of JNK inhibitor SP600125 in combination with nicotinamide and simvastatin protected porcine islets from peritransplant inflammation and apoptosis. Intraductal administration of JNK inhibitor protected islets by inhibiting the production of proinflammatory cytokines IL-
1β, TNFα, IFN-γ, IL-6, IL-8, and MCP-1 in vivo152.
Table 1.1. Antiinflammatory agents in clinical allogenic islet transplantation.
Pub. Pt Induction Maintenance Insulin year no. Anti-inflammatory agents therapy therapy independent Ref 2005 8 Etanercept: 50 mg i.v. rATG Sirolimus 8/8 after 140 pretransplant and 25 mg s.c. on Daclizumab Tacrolimus single days 3, 7, and 10 MMF infusion 2005 8 Infliximab: 5 mg/kg i.v. Daclizumab Sirolimus 7/8 141 pretransplant Tacrolimus 2008 7 Infliximab or etanercept: 5 Daclizumab Sirolimus 6/7 153 mg/kg i.v. pretransplant or 50 Tacrolimus mg i.v. pretransplant and 25 mg (Low-dose s.c. twice weekly for 2 weeks steroids for 3 pts) 2008 3 Etanercept: 50 mg i.v. Alemtuzumab Sirolimus 2/3 154 Tacrolimus pretransplant and 25 mg s.c. MMF twice weekly for 2 weeks 2008 6 Etanercept: 50 mg i.v. rATG (for first Cyclosporine 5/6 155 pretransplant and 24 mg s.c. on transplant) Everolimus days 3, 7, and 10 Daclizumab (for 2nd transplant) 2008 6 Etanercept: 50 mg i.v. Daclizumab Sirolimus 6/6 156 pretransplant and 24 mg s.c. on Tacrolimus days 3, 7 and 10 2011 3 Etanercept and anakinra: 50 mg rATG Tacrolimus 3/3 145 i.v. pretransplant and 25 mg s.c. MMF on days 3, 6, and 10 and 100 mg i.v. pretransplant and 100 mg s.c. for 7 days after transplant 2012* 22 Anti-TNFα T-cell depletion NA 11/22 for 5 142 protocol years i.v. indicates intravenous; II, insulin independence; MMF, mycophenolate mofetil; rATG, rabbit antithymocyte globulin; s.c., subcutaneous; TNF, tumor necrosis factor.
25 Withaferin A
To counter inflammation, one approach we explored involved WA, a naturally
occurring plant-derived molecule. WA has been known for its multifunctional abilities,
including its strong antiinflammatory, antioxidant, and antiangiogenic properties157. WA
is originally derived from a plant called Withania somnifera, which has been used for
centuries as traditional medicine with proven clinical safety158, 159. Several compounds have been extracted from the leaves of Withania somnifera, but a study by Kaileh et al. identified WA as the most potent inhibitor of the transcription factor NFκB based on IκB kinase β (IKKβ) hyperphosphorylation preventing degradation of IκB160. Another group
of researchers suggested that WA prevents activation of NFκB by intercepting the
NEMO/IKKβ complex formation161. WA has been widely investigated as an
antiinflammatory compound in cystic fibrosis and also to treat other chronic
inflammatory diseases such as rheumatoid arthritis, asthma, and inflammatory bowel
disease162-164.
Proinflammatory cytokines such as IL-1β, TNFα, and IFN-γ have been shown to
cause apoptosis of islets by activation of the NFκB pathway165,166. Isolated islets express
tissue factor and chemokines MCP-1 and IL-8, which are regulated mostly by NFκB38.
Therefore, blocking NFκB activation in islets is a potential strategy to improve islet
transplant outcomes. To test this hypothesis, WA was used because of its strong
inhibition of NFκB164.
Using a syngeneic mouse islet transplant model, the effectiveness of WA to
protect transplanted islets from cytokine-mediated damage was demonstrated. Mouse
syngeneic islets were transplanted intraportally and normoglycemia was achieved after
26 WA treatment, but control mice could not reverse diabetes. Islets cultured in cytokines experienced a significant increase in apoptotic cells, which was effectively prevented by
WA pretreatment (Figure 1.7).
a 40 d 30
20
10
0
Blood glucose (mmol/l) 0 10 20 30 Days post transplant b 40 30 Control CYT CYT+0.5WA CYT+1.0WA 20 10 40 0 Blood glucose (mmol/l) 0 10 20 30 + Days post transplant 30 c 40 20 30
20
10 10 Percent TUNELPercent 0 Blood glucose (mmol/l) 0 10 20 30 0 Treatment Days Post Transplant Period Control Cyt Cyt+0.5WA Cyt+1.0WA
Figure 1.7. WA improved outcomes in syngeneic mice and reduced cytokine-induced apoptosis. (a) Intraportal islet transplantation into C57BL/6 mice had a success rate of 100% (3/3) with 400 islets, but (b) using 200 syngeneic islets with injection of 0.5mL PBS (vehicle) resulted in 0% (0/5) of recipients achieving normoglycemia. (c) Daily administration of WA (1.25mg/kg body weight) starting immediately after transplantation and lasting 7 days yielded a much higher success rate of 83% (5/6), (d) Cytokine mediated beta-cell apoptosis was confirmed by TUNEL staining. Human islets were stained for insulin (red) and apoptotic nuclei (green). Manual counting of more than 250 nuclei revealed higher levels of apoptosis when islets were exposed to cytokines, which was significantly reduced by the addition of WA, which returned apoptosis levels to control levels (1.0 μg/ml WA).
NFκB activation induced by cytokines was efficiently blocked by WA pretreatment of islets (Figure 1.8). Pretreatment of islets with WA significantly reduced the production of proinflammatory cytokines and chemokines regulated by NFκB (Figure
1.9).
27 A Control CYT CYT + WA B 6 Control MIN6 5 CYT CYT + WA INS-1 IκB 4 *
Human Islet
IkB:ERK 3
MIN6 Ratio 2
ERK INS-1 1 * * Human Islet 0 MIN6 INS-1 Human Islet
Figure 1.8. Western blot showing IκB degradation upon cytokine treatment but WA prevents it in β cell line and human islets. Immunoblot analysis of IκB protein in human islets and beta-cell lines treated with a cytokine cocktail (CYT) for 20 min in the presence or absence of WA. Experiments were repeated three times and performed in duplicate. (b) Graphical representations are expressed as means ± SD (black- control, grey-cytokine treated, white-cytokine+1μg/ml WA).
a d 8000 8000 IL-8 IP-10 6000 6000 *
4000 * 4000 * pg/Islet pg/ Islet * 2000 2000
0 0
b e 2500 60 MCP-1 MIP-1α 2000 50 40 1500 * 30 / Islet * 1000 * pg
pg/ Islet 20 * 500 10 0 0
c f 800 500 G-CSF IL-6 400 600 * 300 * / Islet 400
pg * 200 * pg/ Islet 200 100 0 0
Figure 1.9. Withaferin A inhibits proinflammatory cytokine/chemokine production by islet treated with cytokines. Quintuplets of fifty handpicked, human islets were cultured for 96h in cytokines or cytokines supplemented with WA at 37oC. Supernatant was collected and analyzed in duplicate with Millipore’s human cytokine detection kit in the Luminex 200®. Results were standardized to the number of islets. Results shown are representative of three independent human islet preparations. Graphical representations are expressed as means ± SD. Significant differences compared to CYT as determined by Turkey-Kramer post-hoc tests are indicated by asterisks (p<0.05).
28
Based on these results, WA was chosen for further investigation of the role of
NFκB inhibition in the alleviation of IBMIR and also to delineate the mechanism that leads to the pathogenesis of CP and to prevent its occurrence.
Future Directions
Inflammation during islet transplantation is multifaceted, and the underlying mechanisms are quite diverse. Several factors could play critical roles in the induction or exacerbation of inflammation. Therefore, it is challenging to identify one particular approach or strategy to eliminate all inflammatory events in islets. A combinatorial approach based on the current understanding of inflammatory mechanisms will likely be required to improve islet transplant outcomes. Overall, isolation of islets from the pancreas with less ischemic time is an important aspect. Furthermore, culturing islets in conditions that are suitable for islet survival and use of antioxidants and antiinflammatory compounds including potent inhibitors of NFκB, which are known to regulate several proinflammatory genes, is important. Since a significant mass of islets are immediately lost to IBMIR, another major focus should be to reduce this response. Although the use of anticoagulants or complement inhibitors shows benefits in in vitro and in vivo settings, they fail to perform effectively in clinical settings. Moreover, in allogenic transplants, the inflammation is severe, and therefore a combined antiinflammatory regimen to reduce ensuing immune response would be required. For instance, LMWDS has shown potential benefits to prevent clotting. This could be combined with antiinflammatory compounds such as anakinra and etanercept and with complement inhibitors to form a complete regimen to block IBMIR. Some of the recent clinical studies, such as use of reparixin to block CXCR1/2 have shown promising outcomes. Several inflammatory mechanisms that
29 cause dysfunction of islets that have been discussed here should be addressed at the clinical level. Future studies should focus on these mechanisms to develop strategies for successful islet transplantation.
30 CHAPTER TWO
Alleviation of Instant Blood-Mediated Inflammatory Reaction in Autologous Conditions through Treatment of Human Islets with Nuclear Factor-Kappa B Inhibitors
This chapter published as: Kanak MA, Takita M, Itoh T, SoRelle JA, Murali S, Kunnathodi F, Shahbazov R, Lawrence MC, Levy MF, Naziruddin B. Alleviation of instant blood-mediated inflammatory reaction in autologous conditions through treatment of human islets with NF-κB inhibitors. Transplantation. 2014;98(5):578-84.
Introduction
Two types of clinical pancreatic islet transplants are currently being performed:
allogeneic islet transplants for those with type 1 diabetes and autologous islet transplants
for patients undergoing total or partial pancreatectomy for refractory chronic
pancreatitis16. After isolation, the islets are generally transfused intraportally, as the liver is considered the preferred site167. A significant number of patients become insulin
independent after transplantation, but this result is transient, and most of these patients
return to using exogenous insulin168. This is a clear indication of poor engraftment and/or
gradual decay of islet function, which could be due to several factors, including poor islet
quality, hypoxia, damage due to instant blood-mediated inflammatory response (IBMIR),
and in the case of allogenic islets, autoimmune and alloimmune responses138, 169.
Previous reports have shown that IBMIR is a significant factor in the damage to
allogenic and xenogenic islet transplants during the peritransplant period52, 54, 72. IBMIR
is a nonspecific response mediated by the innate immune system characterized by
coagulation, complement activation, platelet adhesion, and leukocyte infiltration into the
islets. Tissue factor (TF) expressed by the islets is considered a major trigger for IBMIR
31 leading to platelet activation and release of downstream inflammatory mediators such as
interleukin (IL)-8, monocyte chemotactic protein (MCP)-1, and macrophage migration
inhibitory factor (MIF)52, 170. We recently demonstrated the occurrence of IBMIR in clinical autologous islet transplantation53. IBMIR under autologous conditions was
characterized by strong coagulation and a rise in proinflammatory cytokines with a
minimal impact on complement activation. Analysis of blood samples drawn during
intraportal islet infusion and in the hours immediately afterwards showed significant islet
damage occurring within 3 hours of the beginning of infusion53.
Nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) has long been known to be the major transcription factor for the regulation of various proinflammatory cytokines. IL-1β and tumor necrosis factor α (TNFα) are two major inflammatory cytokines that are released rampantly during IBMIR. These cytokines are regulated by NFκB and can induce transcription of many other downstream products, such as inducible nitric oxide synthases, prostaglandin E2, and interferon gamma- inducible protein 10 (IP-10). In a syngeneic islet transplant model, culturing islets with the NFκB inhibitor dehydroxymethylepoxyquinomicin resulted in improved islet engraftment171. We have also shown that treatment of islets with withaferin A (WA),
which is an NFκB inhibitor, resulted in protection of islets from proinflammatory
cytokine–induced damage and improved survival of syngeneic mouse islets transplanted
intraportally126. Previous reports have also shown that TF expression is also regulated by
NFκB signaling172.
WA is a steroidal lactone isolated from the plant Withania somnifera that has
been reported to have antiinflammatory, antiangiogenic, and anticancer effects157. WA
32 has been used in Southeast Asia and the Middle East for a long time to cure various inflammatory disorders. A recent mechanistic investigation has shown that WA hyperphosphorylates IκB kinase beta and prevents phosphorylation of inhibitor of κB
(IκB) and thereby inhibits NFκB activation161. In the present study, we also employed
CAY10512 (CAY; a potent analog of resveratrol), another known NFκB inhibitor, for further confirmation173.
We used a miniature tube model174 comprising a mixture of pure human pancreatic islets mixed with autologous blood to evaluate the effects of NFκB inhibitors.
We hypothesized that specific inhibition of NFκB can alleviate the inflammatory component of IBMIR and improve the survival of transplanted islets during the crucial peritransplant phase.
Materials and Methods
Human Islet Isolation and Blood Collection
Research-grade human pancreases from brain-dead donors were provided from two local organ procurement organizations (Southwest Transplant Alliance, Dallas, TX, and LifeGift, Houston, TX). Pancreases were procured and preserved with the standard two-layer method. Islets were isolated using the modified Ricordi’s method175, 176. Blood samples from the donors were collected during pancreas procurement in heparinized tubes (BD Biosciences, Franklin Lakes, NJ) to prevent coagulation. Blood samples were maintained in a refrigerator until the start of the experiment (not more than 36 hours).
33
Miniaturized In Vitro Tube Model
Islets were cultured in CMRL 1066 islet culture medium (Mediatech, Manassas,
VA) overnight at 37°C with 5% CO2 to recover from the isolation stress. Prior to the
experiment, islets were washed and counted, 2000 islet equivalents (IEQ) were treated with CMRL medium containing 1.0 ug/mL WA (Enzo Life Sciences, Farmingdale, NY),
0.15 µM CAY (Cayman Chemicals, Ann Arbor, MI), or no drug for 1 hour, and blood samples were allowed to equilibrate at 37°C. The blood was aliquoted in heparinized
Eppendorf tubes (Cardinal Health, Dublin, OH) (0.5 mL in each tube), the islets (~200
IEQs/200uL conditioned media) were added to the blood, and the tubes with blood and
islets were placed in a 37°C shaker incubator. Tubes were removed from the incubator at
their respective time points.
Luminex Multiplex Assay, ELISA, and Viability
The procedures on Luminex multiplex assay and the C-peptide and proinsulin
enzyme-linked immunosorbent assay (ELISA) were performed on serum samples as per
the manufacturer’s protocol. Islets recovered after 6 hours of culture with blood were
washed several times with phosphate-buffered saline to remove red blood cells. Cells were then stained for 10 minutes with Hoechst 33342 and propidium iodide. ImageJ software was used to semiquantitatively determine the percentage viability of islets.
Neutrophil Infiltration in Purified Human Islets Mixed with Autologous Blood
Formalin-fixed paraffin-embedded islet clots were sectioned in 5-micron sections, and polymorphonuclear neutrophil staining was performed using Naphthol AS-D chloroacetate esterase (Sigma Aldrich, St. Louis, MO). At least 15 separate islets were
34
evaluated for neutrophil infiltration. Neutrophil infiltration was classified by counting
polymorphonuclear neutrophils in and around individual islets. Insulin staining was
performed on serial sections using rabbit anti-insulin (Cell Signal, Boston, MA) and anti-
rabbit HRP secondary antibody (Cell Signal, Boston, MA). Color was developed using a
DAB chromogen substrate kit (DAKO, Carpinteria, CA).
Fluorescence Staining for Tissue Factor
Islet tissue sections (5 μm) were obtained after fixing in 10% formalin and
embedding in paraffin. Tissue sections were deparaffinized, and heat-mediated antigen
retrieval was performed. TF was stained with mouse anti-TF antibody (Abcam,
Cambridge, England) following anti-mouse Alexa Flour 594 (Invitrogen, Grand Island,
NY), along with insulin staining with guinea pig anti-insulin antibody (Sigma, St. Louis,
MO) following anti-guinea pig Alexa Flour 488 (Invitrogen, Grand Island, NY) and 4',6- diamidino-2-phenylindole (Sigma-Aldrich, St. Louis, MO).
Statistical Analysis
Data were expressed as mean ± standard error (SE). Data were analyzed using
GraphPad Prism version 6.03 (GraphPad Software, Inc., San Diego, CA). The two-way repeated-measure (RM) analysis of variance (ANOVA) followed by Tukey's or Dunnett post hoc test was performed to evaluate statistical significance between time points or groups. Statistical significance was considered for P values <0.05.
35
Results
Islet Viability after Culture with Autologous Blood
Islets treated with and without WA or CAY were cultured with autologous blood
for 6 hours. In the control group, the viability of islets as analyzed by propidium iodide
and Hoechst 33342 staining was significantly reduced at 6 hours after culture initiation
compared to 1 hour after culture initiation (P < 0.01), but no significant differences in
viability between 1 and 6 hours were seen in the WA and CAY groups (Figure 2.1). In
addition, there were significant differences in 6-hour viability between the control, WA,
and CAY groups (56.4 ± 10.7, 83.8 ± 2.6, and 84.7 ± 4.1 % [average ± SE], respectively;
P < 0.01).
Figure 2.1. Viability of islets after culture in autologous blood. The viability was significantly decreased at 6 hours compared with 1 hour after the culture in the control group (**P < 0.01) but not in either NFκB inhibitor–treated group. When comparing viability at 6 hours, significant differences were found between the control and the withaferin A (WA) and CAY10512 (CAY) groups (**P < 0.01). Data express average ± SE based on 6 independent experiments. P values were calculated by two-way ANOVA with Tukey’s multiple comparison test.
36
Human Islet Damage and the Impact of NFκB Inhibitors
We investigated rapid C-peptide and proinsulin release from islets in the tube model to detect early islet damage. Only the control group showed a significant elevation of C-peptide level in the plasma at 3 and 6 hours compared to baseline (P < 0.05) as well as compared to the WA and CAY groups (P < 0.05, Figure 2.2a). Similarly, only the control group showed a significant increase of the proinsulin level at 1 and 3 hours (P <
0.01) compared to the baseline, although no significant differences between the
Figure 2.2. Autologous islet damage and the impact of NFκB inhibitors. The plasma levels of C-peptide (a), proinsulin (b), and thrombin-antithrombin (TAT) (c) in an autologous islet tube model are shown. Solid line (black circle), solid line (open circle), and dotted lines show the control, withaferin A (WA), and CAY10512 (CAY) groups, respectively. Symbols indicate the P value compared to baseline within each group of control*, WA‡ or CAY# and the significance levels: *P<0.05 and **<0.01. Also, the symbols indicate the P value when comparing the control and WA§ or CAY† groups. Data express average ± SE based on 3 independent experiments. Repeated-measurement two-way ANOVA with Dunnett’s multiple comparison test was performed for statistical evaluation.
37
control and NFκB inhibitor groups were found (Figure 2.2b). TAT levels were
significantly elevated, with a peak at 6 hours in all groups (P < 0.01 between control and
CAY groups; P < 0.05 between control and WA group; Figure 2.2c); there was no significant difference in TAT levels between the three groups. These results were expected because the NFκB signaling pathway is not involved in the formation of the
TAT complex.
Proinflammatory Cytokine Responses
Levels of proinflammatory cytokines in the plasma samples were determined using Millipore multiplex bead assay (Figure 2.3). In the control group, there was a significant elevation in the proinflammatory cytokines TNFα, MCP-1, IL-8, and IL-6 (P
<0.01, 0.01, 0.05, and 0.05, respectively), but not in IP-10, when compared to baseline
(Figure 2.3). Both WA and CAY blocked the significant elevation of cytokine levels up
to 6 hours, except for IL-6 in the WA group.
Neutrophil Infiltration in Autologous Islets in Tube Model
The clots of blood and islets at 3 hours were stained with Naphthol AS-D
chloroacetate esterase to evaluate neutrophil infiltration (Figure 2.4). The number of
infiltrated neutrophils was higher in the islets in the control group (Figure 2.4a). In
contrast, the islet clots obtained from the WA- and CAY-treated groups showed very few to no neutrophils (Figure 2.4b and c). The number of neutrophils in an islet was calculated from 20 different fields. The average of the number of infiltrated neutrophils was significantly lower in both the WA (P <0.0001) and CAY (P < 0.001) groups compared to the control (Figure 2.4d).
38
Figure 2.3. Inhibition of proinflammatory cytokine secretion by NFκB inhibitors. The plasma levels of TNFα (a), MCP-1 (b), IP-10 (c), IL-8 (d), and IL-6 (e) are shown. Solid line (black circle), solid line (open circle), and dotted lines show control, withaferin A (WA), and CAY10512 (CAY) groups, respectively. Symbols indicate the P value compared to baseline within each group of control* or WA‡ and the significance levels: *P<0.05 and **<0.01. Data expressed as average ± SE based on 3 independent experiments. Repeated-measurement two-way ANOVA with Dunnett’s multiple comparison test was performed for statistical evaluation.
39
Figure 2.4. Neutrophil infiltration in islet clots. Neutrophils were stained by Naphthol AS-D chloroacetate esterase reactions (purple), counterstaining with hematoxylin (blue) and insulin (brown). Staining was performed on islet clots obtained after 3 hours of exposure to autologous blood. Representative views of hematoxylin and eosin staining in control (a), insulin staining (b), and neutrophil staining (c) are shown. Neutrophil staining is also shown in control (d), withaferin A–treated (e), and CAY10512 (CAY)–treated (f) groups. Scale bar indicates 50 μm. The number of neutrophils within an islet was counted (g). Data express average ± SE based on 20 counts from each group. **P < 0.01 and ***P < 0.0001 evaluated with ANOVA followed by Tukey’s test.
Tissue Factor Expression in Islets
Islets can express TF, which could potentially trigger coagulation and induce inflammation52. We evaluated TF expression with fluorescence staining in conjunction with insulin using an autologous islet blood sample at 3 hours (Figure 2.5). Strong TF expression was observed in the control group, but not in the WA or CAY groups.
Staining for TF was more intense on insulin-positive cells.
40
Figure 2.5. Tissue factor (TF) expression in islets after exposure to autologous blood. The expression of TF was evaluated with fluorescence stain (red) where nuclei and insulin were stained as blue and green. The autologous islet-blood samples were taken at 3 hours after the culture initiation. The white bar indicates 50 μm. Representative sections are shown. WA indicates withaferin A; CAY, CAY10512.
Discussion
IBMIR is a critical problem in both allogeneic and autologous islet transplantation
that needs to be addressed to improve the outcome of islet transplantation. Nilsson’s
group was the first to describe the characteristics of IBMIR and to extensively investigate
different interventions to block IBMIR53. It has been well established that NFκB is a
major transcription factor involved in the modulation of the inflammatory response177. In
this study, we implemented NFκB blockade as a strategy to reduce the activation of
IBMIR using WA and CAY, which are NFκB inhibitors157, 173. A miniaturized in vitro
41
tube model was employed to investigate the effect of these two drugs on IBMIR174. Our data showed that NFκB inhibitors could be potent agents for pretreatment of islets to ameliorate IBMIR, supported by the significant reduction in proinflammatory cytokine levels in the mixture with autologous blood.
NFκB is one of the key transcription factors activated in response to stress177.
Activation of NFκB, depending on the type of stimuli, could be either protective or
harmful to cells. A gene array study showed that pancreatic islet isolation and the
following culture of islets led to an increase in the mRNA expression of proinflammatory
cytokines in islets178. This increased expression of proinflammatory cytokines might in
turn lead to stress and activation of NFκB in islet cells. Thus, we hypothesized that once
islets are mixed with autologous blood, the innate immune system could activate, and this
could result in secretion of chemokines by the islets, which recruits monocytes and
neutrophils.
After 6-hour culture with autologous blood, viability was significantly preserved
in islets treated with WA and CAY when compared with control (Figure 2.1), suggesting
that WA not only protects islets from IBMIR but also maintains islet viability by other
mechanisms, including prevention of apoptosis. A recent study from our laboratory has
shown that islets cultured in the presence of WA have much better viability compared to
control islets after 48 hours126. The study also showed that inflammatory cytokine–
mediated apoptosis was also reduced in the presence of WA.
Both clinical and in vitro studies have shown that, immediately after islet infusion
or mixing in autologous blood, C-peptide and proinsulin levels rise rapidly, and this
increase could be due to islet damage174. Using our in vitro model, we have also shown
42
that mixing of islets in the blood led to a rapid increase in C-peptide and proinsulin
levels, suggesting islet damage, but when islets were precultured with WA or CAY, the
C-peptide and proinsulin levels were significantly lower, suggesting less islet damage
(Figures 2.2a and 2.2b). It is crucial to prevent the initial damage that occurs to the islets,
because it could lead to a sequence of immunological events eventually affecting the
entire graft. A recent clinical study using positron emission tomography showed that 25%
of islets are lost immediately after transplantation55. Therefore, preventing islet damage
in the initial stages may be the key to successful long-term insulin independence.
With IBMIR, the levels of TAT in plasma increase immediately after the islets
come in contact with blood, indicating the activation of coagulation170. Consistent with
other reports, our data have also shown an immediate increase in TAT levels when islets
were mixed with autologous blood. No influence of WA or CAY on TAT levels was
noticed (Figure 2.2c); however, this was an expected result because the process of
coagulation is not NFκB dependent. On the other hand, it could be argued that
coagulation is triggered by the TF expressed and released by the islets. TF expression is
known to be partly regulated by NFκB172. Islets express high levels of TF, and this interacts with factor VIIa and activates the extrinsic coagulation cascade. Therefore, blocking the activation of NFκB represents a sound strategy to inhibit IBMIR. The TF gene is also regulated by several other transcription factors such as Egr, AP-1, and
NFAT179. Various signals lead to activation of different transcription factors that result in
the induction of the TF gene, and synergistic regulation by several transcription factors
would be required for TF expression. The regulation of the TF gene by NFκB is induced
mainly by cytokine stimulation such as IL-1β, TNFα, and by pathogen-associated
43
molecular patterns such as lipopolysaccharide180. In the case of IBMIR during islet
transplantation, the major factor that could induce TF expression in the islets would be
the proinflammatory cytokines. Previous studies on human monocytes have shown that
preventing IκBα degradation resulted in attenuation of TF gene expression181, and use of
salicylates such as aspirin has similar effects179. Therefore, blocking TF expression by
NFκB would be protective for the islets during early stages of transplantation. We have
shown that both WA and CAY significantly prevent TF expression in islets when
compared with the control. Since we cultured islets in WA or CAY only for an hour
before mixing with autologous blood, a prolonged treatment of NFκB might further
reduce TF expression in islets and prevent the activation of coagulation.
Our recent clinical study showed an increase in the levels of proinflammatory
cytokines and chemokines immediately after infusion of islets into the portal vein174. The
presence of high levels of these cytokines amplifies the inflammatory response, making
the islets more susceptible to cytokine-mediated apoptosis. In our in vitro study, we have demonstrated that the plasma levels of several proinflammatory cytokines were increased after mixing of islets with blood. TNFα is a proinflammatory cytokine known to induce apoptosis in β cells182. We found that TNFα levels increased gradually and reached a maximum at 3 and 6 hours. In both the WA and CAY groups, the level of TNFα did not increase and remained low at all time points (Figure 2.3a). IL-6, which can be either
proinflammatory or antiinflammatory depending on the stimuli, was also found to
increase in control samples, whereas the WA- and CAY-treated samples efficiently
suppressed the secretion of this cytokine (Figure 2.3e). IP-10 levels in the plasma were high to start with and remained high, but in the WA and CAY groups, the level of IP-10
44
was lower compared with the control (Figure 2.3c). IP-10 is considered one of the major
proinflammatory cytokines that can modulate islet graft survival84. MCP-1 is responsible
for attracting monocytes to the site of injury183. When islets are mixed with blood, MCP-
1 levels in the plasma begin to rise and remain high up to 6 hours. When WA- or CAY-
treated islets were mixed with blood, the MCP-1 level did not change (Figure 2.3b),
suggesting prevention of islet damage by macrophages. IL-8 is also a cytokine that
recruits polymorphonuclear cells to the site of inflammation. The level of IL-8 also
increased rapidly after mixing blood and islets, and this elevation was prevented in the
WA and CAY groups (Figure 2.3d). Other key proinflammatory cytokines, IL-1β and
interferon γ, were undetected in the samples.
Our in vitro tube model was validated for the study of islets mixed with blood
174 based on a comprehensive analysis of pH, pO2 and glucose levels . Since our present
study using the in vitro tube model showed that both WA and CAY can inhibit the
secretion of proinflammatory cytokines/chemokines, it is appropriate to show the
resulting outcome. Islet clots were sectioned and stained for neutrophils. The islets that
were mixed with blood showed massive infiltration of neutrophils even at 3 hours,
whereas the WA- and CAY-treated islets showed a very significant reduction in the
number of neutrophils infiltrated per islet (Figure 2.4). Thus, the histology results
correlate with proinflammatory cytokine or chemokine levels, especially with IL-8,
which is required for the maturation and infiltration of neutrophils.
The present study focused on the instant response occurring when purified islets
come into contact with blood. Long-term benefits of treatment with NFκB blockers on
islet graft outcome remain to be determined. IBMIR against allogenic and xenogenic
45 islets may be stronger than against autologous islets due to the involvement of humoral
immune components such as autoantibodies, alloantibodies, and xenoreactive antibodies.
Future studies using this miniature tube model could include analyze the effects of
inhibitors for various components of IBMIR. In summary, we have demonstrated that a
miniaturized in vitro tube model is an effective tool to study IBMIR. We have also shown that blocking NFκB is a promising strategy to alleviate the harmful effects of IBMIR on transplanted islets.
46
CHAPTER THREE
Evaluation of MicroRNA375 as a Novel Biomarker for Graft Damage in Clinical Islet Transplantation
This chapter published as: Kanak MA, Takita M, Shahbazov R, Lawrence MC, Chung WY, Dennison AR, et al. Evaluation of microRNA375 as a novel biomarker for graft damage in clinical islet transplantation. Transplantation. 2015 Mar 12 [Epub ahead of print].
Introduction
Transplantation of allogeneic islets from deceased donors into patients with type 1 diabetes has shown promising outcomes by restoring normoglycemia and eliminating the risk of hypoglycemic episodes139. In addition, for advanced cases of chronic pancreatitis and some benign pancreatic tumors, total pancreatectomy with islet autotransplantation
(TPIAT) offers relief from intractable pain and retention of endocrine function184, 185. In both forms of islet transplantation, a significant mass of islets are lost immediately after infusion into the portal vein of the liver due to an innate immune response called instant blood-mediated inflammatory reaction (IBMIR)51, 53, 56. The process of IBMIR encompasses coagulation, proinflammatory cytokine/chemokine production, complement activation, and infiltration of leukocytes56. In the case of type 1 diabetes, islets engrafted in the liver eventually undergo chronic rejection by the immune system186.
After transplantation, it has been challenging to investigate islet graft survival or chronic rejection in a patient. In clinical practice, islet graft function has been monitored by metabolic function via blood glucose, insulin, and C-peptide levels, as well as through imaging187. However, these tests are influenced by physiological changes, including islet
47 stimulation. It is necessary to identify a biomarker that can specifically detect islet
damage independent of metabolic stimuli.
One option for a biomarker is micro RNAs (miRNAs), a class of small noncoding
RNA (18–24 bp) known to regulate gene expression posttranscriptionally. According to
the miRBase registry (www.mirbase.org), about 2578 mature miRNA sequences have
been identified in the human genome188. Recently, miRNAs present in the circulation
have been found to serve as biomarkers for various cancers and other diseases189, 190. An
islet-specific miR375 reported by Poy et al. is known to regulate insulin secretion by
regulating the protein myotrophin involved in intracellular trafficking191. A more
elaborate study using miR375 knockout mice demonstrated its importance in the
development of β cells192. At the same time, gene array analysis of miRNAs in β cells
showed no changes in the level of miR375 at different glucose concentrations193.
Recently, it was demonstrated that islet-specific miR375 is released in the serum of streptozotocin-injected mice and also in prediabetic NOD mice, both of which are models for β cell destruction; these results suggest that miR375 is released as a result of pancreatic β-cell death194.
Hence, we investigated whether miR375 could be a potential biomarker for islet
damage in clinical islet transplantation. We first used a miniaturized in vitro tube model
to determine if miR375 was released by damaged islets in response to autologous blood
and then measured miR375 using reverse-transcription polymerase chain reaction (RT-
PCR) in serum samples of clinical autologous and allogeneic islet transplant patients.
48 Materials and Methods
Patients
Levels of miR375 were tested in 29 patients: 15 autologous islet recipients (i.e., patients with chronic pancreatitis who underwent TPIAT), 8 allogeneic recipients with type 1 diabetes, and 6 control patients with total pancreatectomy alone; all patients were from Baylor University Medical Center (Dallas, TX, USA) or the University of Leicester
(Leicester, UK). This study was approved by the institutional review board, and written informed consent was obtained from all patients.
For both autologous and allogeneic transplants, pancreases were preserved and processed for islet isolation as described earlier176, 195. The final preparation of islets was assessed for count, purity, viability, and gram stain prior to infusion without any culture.
Isolated islets were infused into the portal vein via the mesenteric vein with heparin
(70 U/kg recipient body weight) over 15–60 min while the patients were under general anesthesia. Beginning 6 h after surgery, patients were continuously infused with 300 U/h heparin for 24 h. Subsequently patients were converted to enoxaparin (Lovenox®;
Sanofi-Aventis, Bridgewater, NJ) at 80 mg/day for 2 weeks. Among the 15 patients who underwent TPIAT, 3 received no antiinflammatory regimen, 6 received etanercept (TNFα blocker) alone, and 6 received a combination of etanercept and anakinra (IL-1β blocker) during islet infusion as described earlier145, 196.
Human research-grade pancreases were used for isolating islets using methods described previously195. Human islets were cultured in Prodo standard islet culture media
(Prodo Labs, Irvine, CA) supplemented with 5% human serum AB and 2% glutamine
(37°C, 5% CO2 up to 96 h).
49
Serum Collection and Storage
Blood samples were obtained for serum and plasma at admission, prior to islet
infusion, during islet infusion, at the completion of islet infusion, and 1 h, 3 h, 6 h, 24 h, 3
days, 5 days, and 7 days after islet infusion. After processing, sera or plasma were
aliquoted in cryogenic tubes and stored at –80°C until further analysis53. Blood drawn
from healthy individuals was used as a control to normalize the patients’ miR375 levels.
Miniaturized In Vitro Tube Model
A recently described in vitro model was used54. Briefly, islets were cultured
overnight to recover from the isolation stress. Before the experiment, islets were washed;
counted and 4000 IEQ were used. Blood samples were allowed to equilibrate at 37°C.
The blood was aliquoted in Eppendorf tubes (Cardinal Health, Dublin, OH) (0.5 mL in
each tube), the islets (~200 IEQ/100 µL media) were added to the blood, and the tubes
with blood and islets were placed in a 37°C shaker incubator. Tubes were removed from
the incubator at their respective time points and centrifuged at 1200 rpm for 5 min to
collect plasma. Another set of tubes where islets were treated with WA was used and
experiments were performed as previously described.
MiRNA Extraction, cDNA Synthesis, and Reverse-Transcription PCR
RNA extraction from 200 μL of samples of serum and/or plasma was performed
using the miRCURY RNA isolation kit for biofluids (Exiqon, Denmark) following the
manufacturer’s protocol. Extracted RNA was treated with rDNase to remove any DNA contamination. Complementary DNA strand synthesis was performed using the miRCURY Universal cDNA synthesis kit (Exiqon, Denmark) following the
50
manufacturer’s protocol. Complementary DNA was diluted 1:40 using nuclease-free
water, and RT-PCR was performed using SYBR green qPCR master mix (Exiqon,
Denmark) and locked nucleic acid–based miRNA primers (Exiqon, Denmark) following
the manufacturer’s protocol. The standard curve was prepared to quantify miRNA using
synthetic miRNA.
Statistical Analysis
All statistical analysis was performed with IBM SPSS Statistics 22 (IBM
Corporation, Armonk, NY) or Prism 6 for Windows (GraphPad Software, Inc., La Jolla,
CA). miR375 expression levels in the in vitro assay and patient serum samples were
normalized with miR16-1-3p and miR423-3p, respectively, using the 2ΔΔCt method197.
The statistical significance of circulating miR375 and serum C-peptide levels for the timeline were evaluated with RM two-way analysis of variance (ANOVA) with
Bonferroni correction followed by the post hoc Games-Howell test, which is designed for unequal variance. Three- or two-group comparison without the timeline was performed with ANOVA with Tukey’s post hoc test or Student t test. Categorical variables were assessed with Pearson chi-square test or Fisher’s exact test. Values are shown as average
± S.E.; P <0.05 was considered statistically significant.
Results
Expression of MiR375 in Human Islets and the Release by Autologous IBMIR
To determine the dose dependency of miR375 in islets, purified human islets were serially diluted. The amount of miR375 in cell lysate exhibited a statistically significant linear regression with islet mass (R2 = 0.94, P < 0.0001) (Figure 3.1a).
51
Using our previously established miniaturized in vitro tube model for investigating IBMIR53, 111. We confirmed the significant elevation of miR375 when islets were mixed with autologous blood, compared with an islet-alone or blood-alone group (P
<0.01 or <0.001, Figure 3.1b). Levels of miR375 peaked immediately after mixing blood with islets and remained high until completion of the experiment (6 hours). Similarly, we found a significant increase in C-peptide levels immediately after mixing islets with blood (Figure 3.1c). The islets alone group showed a short peak of C-peptide secretion at
15 minutes, while no elevation was observed in miR375 level, suggesting that C-peptide can be influenced by mechanical stress, unlike miR375. Of note, 4.46 fmol of miR375 was observed at 6-hour exposure of islets to autologous blood, which corresponds to
39.4% (78.8 out of 200 IEQ) islet damage.
To determine if WA protected islets from IBMIR-mediated damage, serum samples from previous in vitro miniaturized tube experiment were used. Human islets were pretreated with WA before mixing with autologous blood, and serum samples were collected at various time points. The miRNA 375 level in the serum of these samples revealed elevation in the nontreated group that continued to increase with time. WA treatment showed significantly reduced miRNA 375 levels at 1-hour, 3-hour, and 6-hour time points (Figure 3.2a). These data also correlated with the proinsulin and C-peptide levels of the samples (Figure 3.2b, 3.2c). Again, these results suggest that WA protects islets from IBMIR-mediated damage.
52
Figure 3.1. miR375 expression is dependent on islet mass and increases similarly to C-peptide in response to islet damage in a miniaturized in vitro tube model. miR375 amount had an excellent linear regression with islet mass (a) (equation: y = 0.05658x + 0.004165, R2 = 0.94, P < 0.0001). Solid and dotted lines show a regression line and the 95% confidence interval, respectively. Both miR375 expression (b) and C-peptide secretion (c) were higher with the blood and autologous islet combination compared with blood alone or islets alone. ** P with RM two-way ANOVA followed by post hoc Games-Howell test <0.01 and *** P <0.001. All data on miR375 were normalized with miR16-1-3. Results in parts b and c show average ± S.E. Data shown are from three independent experiments.
Circulating MiR375 in Clinical Allogeneic and Autologous Islet Transplantation
When comparing miR375 levels in the 15 autologous islet recipients, 8 allogeneic
recipients, and 6 control patients with total pancreatectomy alone, we found no
significant differences in patient characteristics, except for pretransplant hemoglobin A1c
levels, although transplanted islet yield and dose were significantly higher in patients
with allogeneic transplant than with autologous transplant (P = 0.008 and <0.001, respectively; Table 3.1).
Circulating miR375 levels were significantly elevated following autologous and allogeneic transplants compared with the control group (Figure 3.3a, P = 0.005 and
<0.001 with RM two-way ANOVA). In a post hoc comparison test, significant difference was seen between autologous and allogeneic patients at 3 hours after islet infusion (P <
0.05). The miR375 levels in autologous recipients peaked 1 hour after islet infusion, corresponding to an average of 1.31 × 104 fmol of miR375 released per patient. This 53 indicated that an average of 231,531 out of 441,731 IEQ transplanted (52.4%) were damaged after TPAIT, as calculated from 5.66 × 10-2 fmol/IEQ, shown in Figure 3.1a.
The autologous and allogeneic transplant groups also showed significant elevation in serum C-peptide level when compared with the control (Figure 3.3b, P = 0.003 and
0.047, respectively). Of note, no correlations with statistical significance were found between miR375 relative expression and transplanted islet mass in either autologous or allogeneic transplants (Figure 3.5).
Patients undergoing autologous islet transplants were further divided into three groups based on administration of 1) no antiinflammatory drug, 2) etanercept alone, or 3) a combination of etanercept and anakinra. No significant differences in patient and transplant characteristics were seen between the three groups (Table 3.1).
Table 3.1. Patient and transplant characteristics in autologous recipients for sub-analysis.
Control Single Double group blockage blockage P (n = 3) (n = 6) (n = 6) value a Patient characteristics Age 46.7 ± 5.2 39.0 ± 2.1 35.5 ± 4.9 0.27 Sex: female, n (%) 2 (66.7%) 4 (66.7%) 4 (66.7%) 1.0 Body weight (kg) 64.4 ± 6.0 77.9 ± 9.3 70.4 ± 3.4 0.49 Body mass index (kg/m2) 23.1 ± 1.6 27.4 ± 3.1 24.4 ± 1.2 0.47 Hemoglobin A1c (%) 5.4 ± 0.3 5.7 ± 0.2 5.4 ± 0.2 0.54 Transplantation Islet yield (×103 IEQ) 436 ± 107 392 ± 45 495 ± 86 0.59 Islet dose (×103 IEQ/kg of body 7.0 ± 1.9 5.1 ± 0.9 6.1 ± 0.8 0.49 weight) Tissue volume (mL) 26.0 ± 2.5 8.6 ± 1.2 9.7 ± 2.4 <0.001 Purity (%) 53.3 ± 6.9 58.3 ± 8.7 46.7 ± 15.0 0.76 Viability (%) 97.0 ± 1.2 96.3 ± 0.5 96.6 ± 0.5 0.81
54
a
b
c
Figure 3.2. Islet damage by IBMIR inhibited by WA (a) miRNA 375, (b) C-peptide and (c) proinsulin. All data on miR375 were normalized with miR 423-3p. Results in parts b and c show average ± S.E. Data shown are from three independent experiments. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.
55
Figure 3.3. miRNA levels in vivo indicate immediate islet damage after infusion. Levels of circulating miR375 (a) and serum C-peptide (b) before and after autologous transplantation (n = 15, solid line with white circle), allogeneic transplantation (n = 8, bold dotted line with black circle), or total pancreatectomy alone (n = 6, dotted line with triangle) are shown. The trend of circulating miR375 levels in patients with TPIAT and TP alone (dotted line with triangle, n = 6) is shown in which the autologous islet recipients were divided into three groups: no blockage (solid line with white circle, n = 3), single blockage of TNFα (dotted line with block circle, n = 6) and double blockage of both TNFα and IL-1β (gray bold line with triangle, n = 6) (c and d). X axis indicates time post-islet infusion or surgery. Y axes in C and D are log scale. Data points show average ± S.E.. *P with RM two-way ANOVA < 0.05, **<0.01 and ***<0.001. a In post hoc analysis, a significant difference was observed between autologous and allogeneic recipients at 3 hours after islet infusion.
56
50
40
30
20 C t valu e
10
0 0 2 4 6 8 10 Log of Concentration
Figure 3.4. Standard curve plot for the quantification of miRNA375. Tenfold serial dilutions of synthetic miR375 were used to prepare standard curve. X and Y axes indicate log of miR375 (fmol/L) and threshold cycle (Ct) value corrected with UniSp6 Spike-in miRNA, respectively. The liner regression (y = -3.673x + 42.19) accounts for an excellent R square of 0.98.
a b
2,500 2,500
2,000 2,000
1,500 1,500
1,000 1,000
500 500 miR375 relative expression miR375 relative miR375 relative expression miR375 relative
0 0
200 400 600 800 200 400 600 800 1,000 1,000 3 3 IEQ transplanted (× 10 ) IEQ transplanted (× 10 )
Figure 3.5. miR375 levels posttransplant and transplanted islet mass dot plots show a relationship between miR375 relative expression levels at 1 hour post-islet infusion and transplanted islet mass (IEQ) in autologous (a) and allogeneic (b) islet transplantation. No correlations with statistical significance were found in either type of transplant.
57 Effect of Antiinflammatory Treatment in Circulating MiR375 in Autologous Islet Transplantation
The levels of miR375 in all three groups were significantly elevated immediately
after islet infusion up to 6 h (P = 0.04 with RM two-way ANOVA for time; Figure 3.3c),
although no significant difference was seen between the three groups. After 24 h,
significant differences were observed between the three groups: miR375 levels in the no- blockage control group remained significantly higher for 7 days postinfusion compared with either the single- or double-blockage groups (P = 0.002 and P = 0.001, respectively,
Figure 3.3d). The second peak at 24 h in the no-blockage control group could be due to islet damage induced by proinflammatory cytokines released after transplantation.
Interestingly, both the single- as well as double-blockage group showed significantly reduced levels in this second miR375 peak, suggesting a decrease in islet damage due to inflammation. The second peak of miR375 level in the double-blockage group was smaller than the peak in the single-blockage group, suggesting better protection of islets from dual blockage of TNFα and IL-1β, but statistical significance was not achieved. No significant difference in the insulin independence rate after surgery was seen between the three groups, although one out of three patients achieved insulin independence in the control group, compared with 4 of 6 in the single-blockage group and 5 of 6 in the
double-blockage group.
Discussion
In this study, we have analyzed miR375 as a biomarker to quantify human islets
and detect damage. We used our previously validated miniaturized in vitro tube model for
the study of IBMIR53, 111 and demonstrated a rapid increase in the miR375 levels in the
58
plasma immediately after mixing islets with blood, which was not seen in islet-only or
blood-only controls. The levels of C-peptide mirrored the levels of miR375. These data
indicate that this early damage is most likely related to the inflammatory response by the
innate immune system. Compared with patients undergoing total pancreatectomy only,
there was a significant increase (~1000-fold) in the serum miR375 level in patients
receiving autologous and allogenic islets, suggesting that miR375 is released from islets
undergoing damage due to inflammation.
The linear relationship between the amount of miR375 and the islet mass is a convincing result. It is estimated that 5.66 × 10-2 fmol of miR375 is present per human
IEQ. In an in vitro study with pure islets from a healthy donor, we found linear regression
that intersects at the origin of the graph (Figure 3.1a), suggesting that miR375 can be a
good biomarker to evaluate islet damage, and the slope of linear regression indicates the
amount of miR375 per islet.
Approximately 40% of autologous islets were damaged 6 hours after mixing with
autologous blood in an in vitro tube model. In our previous study, we showed that the
viability of islets was significantly decreased to 56.4% at 6 hours after mixture of
autologous blood and islet111. The amount of damaged islets, which was estimated by
miR375, might reflect the loss of viability. In a previous study, Eriksson et al. estimated
that approximately half of islets were lost within 1 hour after allogeneic islet
transplantation based on changes in radioactivity measured by positron emission
tomography combined with computed tomography (PET/CT)55. Similarly, our clinical
data showed that 52.4% of transplanted islets were damaged after TPIAT. Similarities in
the percentage of damaged islets in allogeneic and autologous transplant suggest that the
59 effects of IBMIR immediately after islet infusion might be equivalent regardless of the
type of transplant, although a validation study with a larger number of patients is
warranted for such a conclusion. The lack of correlation between miR375 levels at 1 hour
posttransplant and islet mass could be explained by differences in donor characteristics
and the degree of islet damage during isolation.
Of note, we found an inverse relationship in the elevation of circulating miR375
and serum C-peptide levels between autologous and allogeneic islet recipients; i.e.,
miR375 levels in autologous patients were higher than those in the allogeneic subjects,
while allogeneic patients showed a higher C-peptide response than autologous patients
(Figure 3.3). This result can be explained by the different biologic characteristics between the two biomarkers. Elevation of C-peptide levels represents both a physiologic response of pancreatic β cells to hyperglycemia and a mechanical release due to islet damage198,
199. In this study, the amount of transplanted islet mass was significantly higher in
allogeneic transplants than in autologous transplants, and thus, greater C-peptide
secretion or release was observed in allogeneic recipients than in autologous patients.
Circulating miR375 has been reported as a specific biomarker for β-cell death and is not
secreted in response to metabolic stimuli194. Autologous recipients undergo major surgery
and are on parenteral nutrition with no metabolic stimulation of transplanted islets. Also,
the autologous islets are isolated from inflamed pancreases and hence could be more
susceptible to immediate damage than allogenic islets184, and in turn, the levels of
circulating miR375 could be higher in autologous transplant than in allogeneic transplant.
Further investigations on biological conditions that modify circulating miR375 levels are
warranted.
60
We then tested the effect of antiinflammatory agents. More elaborate analysis of
miR375 peaks in autologous patients without any antiinflammatory agent demonstrated
an early peak at less than 1 h and a second peak at 24 h, which dropped gradually. On the
other hand, the patients who received antiinflammatory agents such as etanercept or a
combination of etanercept and anakinra had a smaller second peak, suggesting that the
islet damage observed after 24 h may be caused by proinflammatory cytokines. In a
recent preliminary study, Piemonti et al200 showed similar results for miR375 levels in
allogenic islet transplant recipients under three different immunosuppression regimens
and autologous islet recipients undergoing CXCR1/2 inhibitor therapy.
In addition to IBMIR, hypoxia in the liver could also cause early damage to
transplanted islets. A previous report from our group showed that the levels of high-
mobility group box 1 indicated islet damage during the peritransplant period199. The
damage to islets occurring due to inflammatory events after transplantation can be
reduced by using antiinflammatory agents, as we have shown here by the use of
etanercept and/or anakinra (Figure 3.3c).
Some of the major criteria to consider when evaluating a biomarker are
sensitivity, specificity, rapid detection, reproducibility, accuracy, and inexpensiveness.
New technologies such as locked nucleic acid primers and probes for miRNA could
significantly improve the sensitivity of the assay. One other characteristic of miR375 that
stands out is tissue specificity; miR375 is highly expressed only in the islets and
somewhat in neuronal cells, which makes it a very specific biomarker for islets191. Some
of the limitations of this study include (i) the small number of autologous and allogenic islet transplant patients analyzed; (ii) the lack of analysis of a broad repertoire of
61 miRNAs for the same objective; and (iii) analysis of a possible inverse correlation between miRNA levels and posttransplant islet graft function.
In summary, miR375 along with C-peptide and proinsulin in plasma/serum could be considered a stronger and more reliable marker for islet damage in clinical islet transplantation. Furthermore, measurement of miR375 should be performed to assess islet damage due to chronic rejection and may also be useful in detecting early islet damage during the development of type 1 diabetes.
62
CHAPTER FOUR
Delineating the Molecular Pathogenesis of Chronic Pancreatitis and Use of NFκB Inhibitor to Prevent its Progression
Introduction
Chronic pancreatitis (CP) is an inflammatory disease characterized by irreversible
structural alterations due to inflammation, fibrosis, and acinar atrophy. Patients with CP
experience debilitating abdominal pain, exocrine and endocrine pancreatic insufficiency,
and severely compromised quality of life201, 202. Factors known to cause CP are
categorized as toxic (e.g., alcohol, tobacco), genetic (e.g., CFTR mutation, SPINK1
mutation), autoimmune, recurrent and severe acute pancreatitis (AP), obstructive (e.g.,
pancreatic divisum, sphincter of Oddi dysfunction), and idiopathic203, 204. CP increases
the risk of pancreatic adenocarcinoma and type 3C diabetes mellitus and can sometimes
be lethal. CP is usually a result of repeated episodes of AP, which involves intra-acinar
trypsinogen activation, vacuole formation, necrosis, and apoptosis of acinar cells and
severe inflammation205. Currently there is no treatment for CP or a unified approach to
control the progression of the disease.
Several reports have identified the significant role played by nuclear factor kappa
B (NFκB) in the pathogenesis of CP206, but the downstream mechanisms in the
progression of the disease are still unclear. In transgenic mice, tamoxifen-induced
activation of NFκB and/or overexpression of active form of inhibitor of kappa B kinase 2
(IKK)-2 in the pancreatic acinar cell increased the severity of CP207, 208. A high-fat diet– induced CP showed increased NFκB activity to be required for the development of
63
disease209. A cerulein-induced experimental model of pancreatitis has been widely used
to investigate the mechanism of CP210, 211.
Endoplasmic reticulum (ER) stress or unfolded protein response (UPR) is a
cellular mechanism to overcome protein overload and regulate protein folding more
efficiently to regain homeostasis212. Sustained or uncontrolled ER stress leads to
apoptosis of the cell which is mainly regulated by transcription factor C/EBP homologous
protein (CHOP)213, 214. During the pathogenesis of pancreatitis, acinar cell death is prominent and this leads to overwhelming amount of protein production in the remaining acinar cells, which may result in ER stress. ER stress has been identified as an early event in pancreatic injury, and prolonged administration of cerulein to mice results in sustained activation of ER stress leading to CP215.
Cells of the innate immune system (neutrophils, macrophages, monocytes, and dendritic cells) use special receptors such as toll-like receptors (TLRs) and/or NOD-like receptors (NLRs) to sense damage or pathogen invasion. These cells express receptors that recognize ligands during tissue damage called damage-associated molecular patterns
(DAMPs) and during pathogen invasion called pathogen-associated molecular patterns.
NLRP3/NALP3 is an extensively investigated member of the NLR family that is responsible for the formation and activation of an inflammasome complex. NLRP3 oligomerizes and forms a complex with apoptosis associated Speck-like protein (ASC), the inflammasome complex, and then recruits pro-caspase 1 to the caspase recruitment domain (Pycard) present on ASC216. Active caspase-1 is released after cleavage of pro-
caspase-1 by the NLRP3 inflammasome. Subsequently, pro-IL-1β and pro-IL-18 are
processed by caspase-1 to yield active IL-1β and IL-18, which are then released in the
64
inflammatory microenvironment to amplify inflammation. NLRP3 inflammasome has
been reported to play a role in chronic inflammatory diseases such as cryopyrin
associated periodic syndromes, gout, atherosclerosis, and type 2 diabetes.
Withaferin A (WA) is a natural steroidal lactone that has been used in ancient
ayurvedic medicine as an anti-inflammatory drug. It has been shown to inhibit NFκB
activation by binding to I kappa kinase β (IKKβ) and preventing phosphorylation of
inhibitor of kappa B (IκB)217. In this study, we have investigated the role of ER stress and
inflammasome in a cerulein-induced CP model. Furthermore, we used WA to investigate
whether blocking NFκB would alleviate pancreatitis.
Materials and Methods
Mice
All mice were housed in filter-topped shoebox cages with autoclaved food and water. C57BL/6 mice purchased from Jackson Laboratories were used throughout the study and all experiments were conducted on age- and sex-matched littermates. Mice
were randomly assigned to control and experimental groups. All experiments were
performed in accordance to Baylor Research Institute’s animal care and use committee guidelines and regulations.
Reagents
Cerulein was purchased from Sigma Aldrich (St. Louis, MO), and WA from Enzo
Life Sciences (Farmingdale, NY). The amylase activity kit, Picrosirius red staining kit, and anti-neutrophil and anti-Ki-67 antibody were obtained from Abcam (Cambridge,
MA). Mouse CD45, CD11b, CD14, and Ly6g antibody were purchased from BioLegend
65
(San Diego, CA). Anti-NFκB antibody was bought from Santa-Cruz Biotechnology
(Dallas, TX). RNAlater and TRIzol were purchased from Life Technologies (Carlsbad,
CA). The high-capacity cDNA reverse transcription kit was obtained from Applied
Biosystems (Waltham, MA), and the qRT-PCR SYBR green/ROX master mix and custom-made human inflammatory gene array kit from Qiagen (Valencia, CA). IL-6 and
MCP-1 Luminex multiplex kit was obtained from Millipore (Darmstadt, Germany).
Acute Pancreatitis
Animals fasted overnight before induction of pancreatitis by cerulein; water was given ad libitum. Mice were injected with cerulein (50 μg/kg intraperitoneally) or saline every hour for a total of 7 injections and were sacrificed 1 hour after the last injection.
WA (1.25 mg/kg) was administered 1 hour before the first cerulein injection (n = 5 mice/group) (Figure 4.1a). Blood was collected from the inferior vena cava for analysis of serum markers of pancreatitis, and pancreas tissue was harvested immediately afterwards. The pancreas was divided for immunohistochemistry, RNA extraction, protein extraction, and immune cell infiltration analysis.
Chronic Pancreatitis
Animals were subjected to repeated episodes of AP by a supramaximal dose of cerulein. The mice fasted overnight before cerulein injection, with an ad libitum supply of water. In the preventive model, CP was induced when cerulein was injected intraperitoneally (50 μg/kg) every hour for a total of 7 injections. This injection was repeated once a week for 4 weeks. Control mice were injected with saline as a sham treatment. Another group of mice received WA (1.25 mg/kg) 1 hour before the first
66
cerulein injection every week (n = 6 mice/group) (Figure 4.1b). In the therapeutic model,
CP was induced when cerulein was injected intraperitoneally (50 μg/kg) every hour for a
total of 6 injections. This injection was repeated twice a week (Mondays and Thursdays)
for 8 weeks. WA (0.625 mg/kg) was injected 1 hour before the first cerulein injection, but
administration was initiated at week 5 and continued until the end of the experiment
(Figure 4.1c). In both models, mice were then sacrificed and tissue was harvested 1 day
after the last cerulein injection.
a. Acute Pancreatitis WA (1.25mg/kg) Weeks 1 Cerulein (50ug/kg) Once per week, 7 hourly shots i.p. b . Chronic Pancreatitis (Preventive) WA (1.25mg/kg) Weeks 1 2 3 4 Cerulein (50ug/kg) Once per week, 7 hourly shots i.p. c. Stringent Chronic Pancreatitis (Therapeutic) WA (0.625mg/kg) Weeks 1 2 3 4 5 6 7 8 Cerulein (50ug/kg) Once per week, 7 hourly shots i.p.
Figure 4.1. Protocol for development and treatment of pancreatitis: (a) acute pancreatitis, (b) chronic pancreatitis (preventive approach), (c) Stringent chronic pancreatitis (therapeutic approach).
Amylase Activity Assay
Serum samples obtained from mice after treatment were harvested and stored at –
80°C until further analysis. Serum was analyzed for amylase activity as per the
manufacturer’s protocol.
67 Histology and Immunohistochemistry
Pancreas tissue was placed in 10% formalin immediately after harvesting and
fixed for at least 48 hours at 4°C before processing. Tissue was embedded in paraffin,
and 5-um sections were made and mounted on slides for staining. Hematoxylin and eosin
staining was performed for histopathological evaluation, and a pathological observer
blinded to the study groups scored the tissue from 0 (normal) to 5 (severely inflamed).
Sirius red staining was performed according to the manufacturer’s protocol on the
sections to evaluate fibrotic change. The sections were deparaffinized in xylene and
rehydrated using ethanol. The slides were incubated in a citrate buffer of pH 6 at 97°C for
20 minutes. Following the antigen retrieval process, the slides were carefully washed 3
times for 5 minutes in Tris-buffered saline (TBS) containing 0.025% Triton X-100. The
slides were blocked in 1% bovine serum albumin (BSA) in TBS for 2 hours at room
temperature to block nonspecific binding of the antibodies. Then the slides were
incubated overnight with primary antibodies, which were diluted in TBS with 1% BSA.
The following day, the slides were gently rinsed 3 times for 5 minutes in TBS 0.025%
Triton X-100. Tetramethylrhodamine- or fluorescein isothiocyanate–labeled secondary
antibodies from Invitrogen were diluted in TBS with 1% BSA. The slides were
counterstained with 4',6-diamidino-2-phenylindole (DAPI) and mounted for microscopic analysis. ImageJ software was used to perform semiquantitative analysis of the staining.
Immune Cell Infiltration Assay
A cell suspension was prepared from the pancreas tissue after digestion with collagenase (2 mg/mL) at 37°C for 15 minutes. Tissue was further mechanically digested by pipetting several times. The suspension was then washed and filtered to obtain only
68
the single cell fraction. The cell suspension obtained was used for flow cytometric
analysis of infiltrated immune cells. 1 × 106 pancreatic cells were labeled with anti-
CD45, anti-CD11b, anti-CD14, and anti-Ly6g antibodies. Stained cells were detected
using FACScanto II, and data were analyzed using FlowJo software.
Luminex Assay
The mouse pancreas was homogenized, and the total lysate concentration was
measured. Lysate was analyzed directly according to the manufacturer’s protocol, and
results were normalized to total lysate concentration.
qRT-PCR
Pancreas tissue was stored in RNAlater at –80°C until the extraction procedure.
Total RNA from the pancreas tissue was extracted using TRIzol reagent. Tissue was
homogenized, and debris was removed by centrifugation. Chloroform was added to
separate the RNA from other biomolecules and was further precipitated using
isopropanol. The RNA pellet was washed with ethanol and quantified using NanoDrop
2000. cDNA synthesis was performed using a high-capacity cDNA reverse transcription
kit as per the manufacturer’s protocol. PCR primers for 18s rRNA, PERK, Atf6, Ern1,
Eif2a, Atf4, Xbp1, CHOP, HMGB1, Pycard, Nlrp3, IL-18, IL-1β, IL-6, TNFα, Nos2,
Casp3, Casp7, and Bax were purchased from Qiagen. qRT-PCR was performed using the
SYBR green/ROX master mix from Qiagen as per the manufacturer’s protocol.
Gene Array
Healthy human pancreas tissue was obtained from cadaveric donors. CP tissue was obtained from patients undergoing total pancreatectomy auto islet transplantation
69 who consented to provide samples for a research study. All protocols were performed according to regulations of Baylor Research Institute’s institutional review board.
Pancreas tissue was used for RNA extraction by TRIzol and cDNA synthesis, as previously described. A custom PCR array for inflammatory and NFκB regulated genes was obtained from Qiagen, with performance and analysis as per the manufacturer’s recommendations.
Statistical Analysis
Statistical analysis was performed using Graphpad Prism 6 software (La Jolla,
CA). Statistical significance for more than two groups was determined by one-way analysis of variance and Tukey’s multiple comparison test. Statistical analysis of two groups was determined using unpaired Student’s t test. Differences were considered significant when P values were less than 0.05.
Results
Effect of WA on Acute Pancreatitis Model
Administration of the cholecystokinin analog cerulein has been widely established as a model for induction of acute and CP218, 219. AP pancreatitis was induced in C57BL/6 mice by 7 hourly intraperitoneal injections of cerulein (50 μg/kg) (Figure 4.1a). The mice were euthanized 1 hour after the last injection to harvest plasma and pancreas tissue for analysis. The incidence of AP was determined by analysis of serum amylase levels and histopathological examination of the pancreatic sections. Cerulein treatment increased serum amylase levels by almost 4-fold compared with controls.
70
To test the ability of WA to inhibit cerulein-induced CP, WA was administered 1 hour prior to cerulein injection. WA treatment significantly reduced cerulein-induced serum amylase elevation (Figure 4.2e). Histopathological examination of tissue revealed significant necrosis of the pancreatic parenchyma and increased infiltration of inflammatory cells in cerulein-treated mice (Figure 4.2a, 4.2b). Administration of WA prevented damage to the pancreatic parenchyma and also reduced inflammatory cell infiltration significantly. Pancreas tissue was digested using collagenase, and single cells were stained for innate immune cell markers (CD45, CD11b, CD14, Ly-6g). Flow cytometric analysis revealed a significant increase in the percentage of leucocytes present in the pancreas 1 hour after the last cerulein injection compared with controls, but WA reduced the percentage of infiltrating leucocytes (Figure 4.2c, 4.2d). Myeloid cells, neutrophils, and macrophages were found to be increased in the mouse pancreas after cerulein administration, but this increase was abolished by WA pretreatment.
Several mechanisms of cerulein-induced pancreatitis have been described, but reactive oxygen species (ROS)–modulated secretion of enzymes by acinar cells has been widely investigated220. ROS activation by cerulein on the AR42J pancreatic acinar cell was not reduced by WA (data not shown). ROS activation is upstream of IKKβ activation220, 221, a known mechanism of inhibition of NFκB activation by WA.
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Figure 4.2. Withaferin A (WA) reduces the severity of acute pancreatitis (AP) and abolishes immune cell infiltration. (a) Hematoxylin and eosin staining of pancreas sections from control, cerulein-treated, and cerulein-plus-WA–treated mice. Representative sections of pancreases from at least 5 mice per group. (b) A histological score of 0 to 5 was assigned by an individual blinded to the study groups, where 0 represents healthy pancreas morphology and 5 represents severe pancreatitis morphology, demonstrating immune cell infiltration, acinar atrophy, intraacinar vacuolization, and/or hemorrhage. At least 4 different loci per pancreas were visually inspected and scored; data are represented as mean ± SD of 5 independent experiments. (c) Flow cytometric analysis of pancreatic cells was performed to investigate immune cell infiltration. Pancreatic cells were stained with CD45, CD11b, Ly6G, and CD14. Data are representative of 5 independent experiments. (d) Representative bar graph showing percentage of CD45-positive leucocytes infiltrating the pancreas. Data are expressed as mean ± SD of 5 mice per group. (e) Elevated amylase level in the serum of mice is a hallmark of AP. Serum amylase was measured using ELISA. Data are expressed as mean ± SD of 5 independent experiments. (b, d, e) *P < 0.05, ***P < 0.001, ****P < 0.0001 (one-way ANOVA with multiple comparison).
72
Attenuation of CP Progression by WA
Two different models of CP were investigated, a preventive and a therapeutic
model. In the preventive model, CP was induced by 7 hourly injections of cerulein (50
μg/kg) injected once a week for 4 weeks. In the treatment group, WA was administered 1
hour prior to cerulein injections every week (Figure 4.1b). Vehicle-treated control mice
exhibited a normal pancreatic morphology and WA administration alone did not cause
any structural change in the pancreatic tissue compared to control (Figure 4.3a). The WA
dose (1.25 mg/kg) was very well tolerated by mice, and there was no significant change
in body weight. CP induced by repeated episodes of AP caused significant morphological
change in the pancreatic parenchyma, as evidenced by hematoxylin and eosin staining of
pancreas tissue. The acinar tissue was atrophic with significant infiltration of
inflammatory cells, ductal metaplasia, and hemorrhage.
Administration of WA 1 hour prior to cerulein injection significantly abolished
progression of CP. Histological examination confirmed that WA protected the pancreatic
parenchyma from cerulein-induced damage and maintained pancreas morphology.
Inflammatory cell infiltration, ductal metaplasia, and hemorrhage were reduced or completely absent in the WA-treated group (Figure 4.3b).
Intraacinar vacuolization is a histological feature in CP and manual blinded
counting revealed a higher number of cells showing intra-acinar vacuolization in the
cerulein-treated group than in the WA group (Figure 4.3c). Blood was collected from mice 1 hour after the last set of cerulein injection. Compared with the control and WA- only–treated group, there was significant elevation of amylase in the serum due to damaged acinar cells leaking enzymes in the blood stream. WA pretreatment reduced the
73
serum amylase levels suggesting a significant reduction in acinar damage due to cerulein
stimulation (Figure 4.3d).
Pancreas tissue collected at the time of harvesting was used to measure local
inflammation by measuring proinflammatory cytokine (IL-6) and chemokine (MCP-1)
from homogenate. Protein levels of both IL-6 and MCP-1 were increased by cerulein injection and WA treatment restored both proteins back to control levels (Figure 4.3e,
4.3f). Together these data suggest that WA has a preventive effect on CP progression.
Presently, there is no prognosis for CP unless it occurs due to heredity or genetic
predisposition11. CP is diagnosed only after several attacks or episodes of AP. Therefore,
to make the experiment clinically relevant, the therapeutic potential of WA was tested
using a therapeutic model. CP was induced by 6 hourly injections of cerulein (50 ug/kg)
injected twice a week for 8 weeks. Mice in the WA treatment group were injected with
cerulein for the first 4 weeks, and WA (0.625 mg/kg) administration was initiated from
week 5 until the end of the procedure (Figure 4.1c). The histopathological examination of
the tissue revealed a normal morphology of vehicle-treated mouse pancreases, while
cerulein administration based on the therapeutic protocol caused pronounced destruction
of pancreatic parenchyma, which was efficiently attenuated by WA administration
(Figure 4.4a). The acinar cell number dropped significantly in cerulein-treated mice
pancreases, and WA treatment protected cells from further damage by cerulein.
One of the hallmarks of advanced CP is the replacement of pancreatic tissue by
fibrosis. To visualize fibrotic changes, pancreas sections were stained for collagen fibers
with Sirius red. Mice injected with cerulein alone demonstrated distinct fibrotic change in
compared with both control and WA-administered mice (Figure 4.4a). The inflammation
74
in the tissue was scored histologically by a blinded observer, and the WA treatment group had a significantly lower score than the cerulein group (Figure 4.4b).
Another hallmark of advanced CP is the lack of enzyme production. Pancreatic amylase after cerulein treatment was undetectable in the serum (data not shown).
Homogenates from the pancreas were used to measure levels of proinflammatory molecules IL-6 and MCP-1. Cerulein treatment significantly increased levels of IL-6 and
MCP-1, which was diminished by WA administration (Figure 4.4c, 4.4d).
Cytoprotective and Antiinflammatory Actions of WA
Cerulein-induced necrotic damage to acinar cell triggers a regenerative response
and proliferation of cells to repair and replace dead cells. Ki67, a marker for proliferation,
was used to assess acinar cell damage. There were little to no Ki67-positive cells in the
pancreas of control mice, but repeated cerulein injections resulted in a significant
increase of Ki67-positive cells in the pancreas (Figure 4.5a). With WA administration,
the percentage of Ki67-positive cells was significantly lower (Figure 4.5b). Comparison
of the preventive and therapeutic CP model revealed more pronounced pancreas damage
in the therapeutic model, but WA was able to potently alleviate the damage caused even
in this model (Figure 4.5c-d).
Tissue sections of mice pancreas were stained for infiltration of neutrophils after
CP was induced. Cerulein-injected mice demonstrated a plethora of granulocytes
infiltrating the pancreas, but WA pretreatment significantly reduced infiltration of
neutrophils (Figure 4.5e, 4.5f).
75
Figure 4.3. Withaferin A (WA) can prevent development of Chronic Pancreatitis (CP). (a) Hematoxylin and eosin staining of pancreas from control, cerulein only, WA only, or cerulein plus WA–treated mice. Representative sections of pancreas from at least 6 mice per group. (b) Histological score was evaluated as previously mentioned from at least 5 different loci per pancreas, with a total of 6 independent experiments per group. (c) Number of intraacinar vacuolizations per frame was manually counted in at least 5 different frames per pancreas and in a total of 6 mice per group. (d) Serum amylase was measured by ELISA after 4 weeks of cerulein injection. Data are expressed as mean ± SD of six independent experiments. Luminex analysis of (e) IL-6 and (f) MCP-1 present in the pancreas homogenate was performed and data were normalized to total protein concentration in the lysate. Data are expressed as mean ± SD of 6 independent experiments. (b–f) *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (one-way ANOVA with multiple comparison).
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Figure 4.4. Withaferin A (WA) can abrogate progression of severe chronic pancreatitis (CP)-therapeutic model. (a) Hematoxylin and eosin staining of pancreas from control, cerulein only, or cerulein plus WA– treated mice. Representative sections of pancreas from at least 6 mice per group. Fibrosis is a hallmark of severe CP, and Sirius red staining was used to visualize fibrotic change in the pancreatic parenchyma. Images are representative of 6 different experiments. (b) Histological score was evaluated as previously mentioned from at least 5 different loci per pancreas, with a total of 6 independent experiments per group. Luminex analysis of (c) IL-6 and (d)) MCP-1 present in the pancreas homogenate was performed and data were normalized to total protein concentration in the lysate. Data are expressed as mean ± SD of 6 independent experiments. (b–d) **P < 0.01, ****P < 0.0001 (one-way ANOVA with multiple comparison).
77
Figure 4.5. Withaferin A (WA) reduces cerulein-induced acinar damage and blocks neutrophil infiltration. (a, c) Immunohistochemical staining of mice pancreas tissue for Ki67, a marker for proliferation, demonstrates severity of damage. DAPI was used to stain the nucleus blue and anti-Ki67 antibody with anti-rabbit fluorescein isothiocyanate–labeled secondary antibody was used. (b, d) ImageJ software was used to determine the percentage of Ki67-positive cells per frame. Data are expressed as mean ± SD representative of 3 different loci in 3 independent experiments. (e) Neutrophil infiltration into the pancreas was visualized by staining for granulocyte marker Ly6G and TRITC-labeled secondary antibody; DAPI was used to stain the nucleus. (f) ImageJ software was used to determine the percentage of neutrophils per frame. Data are expressed as mean ± SD representative of 3 different loci in 3 independent experiments. **P < 0.01, ***P < 0.001 (one-way ANOVA with multiple comparison).
78
Neutrophils, one of the first responder cells to the site of injury, initiate the
inflammatory cascade207, 208, 222. Thus, lack of neutrophil infiltration in WA-pretreated mice suggests reduced inflammation. Furthermore, WA successfully reduced mRNA expression of proinflammatory cytokines (IL-6, TNFa) and proapoptotic molecules
(iNOS, Caspase 3, Caspase 7, and Bax) that were upregulated by cerulein administration
(Figure 4.6b–4.6g). In the therapeutic model, inhibition of IL-6, TNFα, Nos2, and Bax by
WA was observed in a similar fashion (Figure 4.7b–4.7e). Together, these data provide substantial evidence of the antiinflammatory and protective role of WA against cerulein-
induced CP.
Ability of WA to Block NFκB Activation and Inhibit ERK Activation
Overexpression or sustained activation of NFκB in the acinar cells increases the severity of cerulein induced pancreatitis207, 208, 222. WA is known to bind to IKKβ and
inhibit phosphorylation of IκBα, hence preventing activation and translocation of NFκB
into the nucleus. Cerulein administration to mice resulted in activation and translocation
of NFκB in the pancreatic acinar cells (Figure 4.6a). WA treatment not only reduced the
expression but also prevented translocation of the active subunit of NFκB into the
nucleus. Extracellular signaling–regulated kinase 1/2 (ERK1/2) has also been shown to
be activated upon cerulein treatment in acinar cells223. The actual signaling sequence is
still not well understood, but can be either upstream or downstream of NFκB activation
or even independent of NFκB signaling. We treated AR42J cell lines with cerulein 100
nM in the presence or absence of WA and found that ERK phosphorylation was
significantly increased by cerulein within 30 minutes. Strikingly, WA treatment
79 significantly inhibited ERK phosphorylation in the groups both with and without cerulein treatment (Figure 4.7a).
Figure 4.6. Withaferin A (WA) inhibits cerulein-induced nuclear factor kappa B (NFκB) in acinar cells and downregulates expression of proinflammatory and proapoptotic molecules. (a) NFκB translocation into the nucleus was visualized by staining the nucleus with DAPI (blue) and staining the P65 subunit of NFκB with anti-P65 and fluorescein isothiocyanate labeled secondary antibody (green). The black arrows show the translocation of NFκB into the nucleus visualized by merging of green and blue. (b–g) Real-time PCR analysis of proinflammatory cytokines and proapoptotic signals was performed on RNA extracted from the pancreases of CP-induced mice and control mice. (b) IL-6, (c) TNFα, (d) Nos2, (e) Casp3, (f) Casp7, (g) Bax. Data are expressed as box and whisker plot, where the top and bottom ends of the box indicate range, the whiskers indicate SD, and the line in the middle indicates median. Data are representative of at least 6 mice per group. *P < 0.05, **P < 0.01 (one-way ANOVA with multiple comparison).
80
Figure 4.7. Withaferin A (WA) downregulates cerulein-induced expression of proinflammatory and proapoptotic molecules in therapeutic CP model. (a) Western blot for total and phosphorylated extracellular signaling–regulated kinase (ERK) of the MAP kinase pathway from the lysates of AR42J cells treated with cerulein (10-7M) for 30 minutes in the presence or absence of WA (10-6M). Data are representative of 3 independent experiments. (b-e) Real-time PCR analysis of proinflammatory cytokines and proapoptotic signals was performed on RNA extracted from the pancreases of cerulein and cerulein+WA treated mice. (b) IL-6, (c) TNFα, (d) Nos2, (e) Bax. Data are expressed as box and whisker plot, where the top and bottom ends of the box indicate range, the whiskers indicate SD, and the line in the middle indicates median. Data are representative of at least 6 mice per group. *P < 0.05, **P < 0.01 (one- way ANOVA with multiple comparison).
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Inhibition of ER Stress Pathway During Pathogenesis of CP
Previous reports have demonstrated the significance of sustained activation of ER stress in the pathogenesis of CP215. Due to the high ER volume and protein load on acinar cells, they are highly susceptible to ER stress224. ER stress leads to overexpression of proteins, such as PERK, ATF6 and IRE1α, in the ER lumen. Upon administration of cerulein, pancreas tissue showed significant upregulation of these ER stress proteins
(Figure 4.8a–4.8c). WA-treated mice pancreases had lower expression of these ER stress proteins, and the downregulation correlated with the reduced damage to the pancreas tissue. RNA expression of downstream signaling molecules in the ER stress pathway was also upregulated during CP induction, which was significantly abolished by WA administration (Figure 4.8d–4.8f). Similar results were observed in the therapeutic model of CP (Figure 4.10a–4.10f).
CHOP induction during ER stress in most cases leads to cell apoptosis225. Upon continuous injection of cerulein, CHOP expression increased significantly, which may result in apoptosis of the acinar cells. Cerulein-induced CHOP expression was reduced in mice pancreases by WA (Figure 4.8g, 4.6h).
Inflammasome Signaling in CP and Attenuation by WA
Inflammasome is a large multimeric protein complex produced and assembled by myeloid cells in many chronic inflammatory diseases226. The formation of inflammasome and its role in CP have not been investigated. Tissue damage results in release of several damage markers of DAMPs, such as high-mobility group box (HMGB-1), heat shock proteins (HSPs), dsDNA, and ATP. Cerulein injection caused a significant increase in the
82 expression of HMGB-1 in the acinar cells, but WA administration significantly reduced this expression (Figure 4.9a).
Figure 4.8. Withaferin A (WA) reduces endoplasmic reticulum (ER) stress. Real-time PCR analysis of (a–f) ER stress regulated genes was performed on RNA extracted from pancreases of CP-induced mice and control mice. Data are expressed as box and whisker plot, where the top and bottom ends of the box indicate range, the whiskers indicate SD, and the line in the middle indicates median. Data are representative of at least 6 mice per group. (a) PERK, (b) Atf6, (c) ERN1, (d) Atf4, (e) Xbp1, (f) CHOP. (g) Western blot analysis of lysates from pancreases of severe CP–induced mice and control mice. (h) Semiquantitative analysis of CHOP protein in the lysates. Data are expressed as mean ± SD of 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (one-way ANOVA with multiple comparison).
83
Figure 4.9. Withaferin A (WA) reduces expression of inflammasome related genes. Real-time PCR analysis inflammasome-related genes was performed on RNA extracted from pancreases of CP-induced mice and control mice. Data are expressed as box and whisker plot, where the top and bottom ends of the box indicate range, the whiskers indicate SD, and the line in the middle indicates median. Data are representative of at least 6 mice per group. (a) HMGB-1, (b) Pycard, (c) NLRP3, (d) IL-18, (e) IL-1β. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (one-way ANOVA with multiple comparison).
Infiltrating immune cells such as macrophages, neutrophils, or dendritic cells can
be primed by the DAMPs and initiate production and activation of inflammasome
molecules. CP induction by cerulein significantly increased the expression levels of
NLRP3, and this expression was blocked when WA was injected into the mice (Figure
4.9c). Another important protein required for activation of inflammasome complex is
ASC, and it has a caspase-binding domain called CARD. Expression of ASC was also
significantly increased when cerulein was injected to induce CP but was efficiently
inhibited by WA (Figure 4.9b). Upon activation of inflammasome, the inactive caspase-1
was cleaved to become active caspase-1, and the activated caspase-1 is required for
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processing and release of IL-1β and IL-18. The expression level of these two cytokines
increased in cerulein-treated mice, but not in WA-pretreated mice (Figure 4.9d, 4.9e).
Similar results were obtained when WA was administered after CP was established in the
therapeutic model (Figure 4.10g–4.10i). In summary, inflammasome signaling took place
during CP, which had not been described previously, and WA administration effectively
inhibited this pathway.
To corroborate the above findings using the cerulein-induced CP model as
relevant to a clinical setting, we tested pancreatic tissue collected from healthy patients
(deceased organ donors) and CP patients (undergoing total pancreatectomy with islet
autotransplantation) for proinflammatory gene expression by RT-PCR array. As shown in
Figure 4.11, most of the proinflammatory genes were highly upregulated (5- to 60-fold
induction) in pancreases from CP patients compared with those from healthy donors.
Discussion
Pancreatitis is characterized by inflammation and parenchymal cell death. These
two key pathologic parameters determine the severity of the disease227. The mechanism of initial insult that leads to pancreatitis is still not clear, although the damage of acinar cells leads to release of several proinflammatory mediators that activate a cascade of molecular signaling and inflammatory events228, 229. In most cases, the inflammation
subsides and results in recovery of the pancreas; this is called AP. In other cases, there is
persistent chronic inflammation that causes continual damage of the pancreas, or there is
recurrent AP, and both situations lead to CP. One of the hallmarks of severe CP is the irreversible replacement of pancreatic acinar cells by fibrotic tissue.
85
Figure 4.10. Real-time PCR analysis of (a–f) ER stress-regulated genes and (g–i) inflammasome-related genes, performed on RNA extracted from pancreases of therapeutic CP-induced mice. Data are expressed as box and whisker plot, where the top and bottom ends of the box indicate range, the whiskers indicate SD, and the line in the middle indicates median. Percent reduction of gene expression by WA compared to cerulein only is shown. Data represent at least 4 mice per group. (a) PERK, (b) Atf6, (c) Ern1, (d) Atf4, (e) Xbp1, (f) CHOP, (g) HMGB1, (h) NLRP3, and (i) IL-1β. NS, not significant, *P<0.05, **P<0.01 (one-way ANOVA with multiple comparison test).
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Healthy Chronic Pancreatitis
Figure 4.11. Proinflammatory and proapoptotic genes upregulated in the pancreas of human CP patients. (a) Heat map showing upregulation of proinflammatory and proapoptotic genes in the pancreases of patients with advanced-stage chronic pancreatitis (6 per group). Pancreas tissue from healthy individuals was used as a control to determine fold change. (b) The table shows genes that were upregulated 5-fold or more in the CP patient tissue compared to control and that were identified as statistically significant changes among the genes analyzed in the array. Analysis and statistics were performed by Sabiosciences-provided software for custom array plates.
In most severe cases, the CP progresses to diabetes or in some instances to
pancreatic adenocarcinoma. Therefore, it is crucial to identify the trigger and mechanism
that causes this life-threatening illness and to develop a cure for the disease. Many
different experimental models have been devised to understand the mechanism that leads
to different types of pancreatitis230. Cerulein-induced pancreatitis has been widely accepted as a clinically relevant model for recurrent acute necrotizing idiopathic pancreatitis. Several reports accrued over the past years have implicated the role of NFκB activation in the pathogenesis of CP206, 207, 210, but other reports have been
87 contradictory231, 232. Gene knockout studies revealed that the absence of NFκB activity
reduced the severity of AP upon cerulein injection210. When IKKβ was made
constitutively active in the acinar cells of mice, cerulein administration worsened the
disease. To further bolster this result, overexpression of dominant negative IKKβ
ameliorated CP208. Another report showed worsening of pancreatitis when RelA was knocked down in the acinar cells of mice231. Gukovsky et al. have reviewed the role of
NFκB in pancreatitis, providing possible theories behind such contradictory reports233.
Nevertheless, the cerulein model has been shown to activate NFκB in the acinar cells, with continuous stimulation leading to CP in mice211. Therefore, we hypothesized that inhibition of NFκB would protect the mice from progressing to CP.
Since AP is the early event that initiates an inflammatory response that may lead
to CP, it is crucial to block it early. In this study, using a cerulein-induced AP model, we
identified that using WA (an inhibitor of NFκB) reduced disease severity and
inflammation. We then tested if WA could protect the mice from recurrent episodes of
AP that would eventually lead to CP. In the first set of experiments, there were striking
histopathological differences between WA- and cerulein-treated mice pancreases
compared with pancreases of mice receiving cerulein injections alone. Serum amylase
levels, immune cell infiltration, Ki-67 staining, and inflammatory cytokine levels in the pancreas were close to control levels in the WA treatment group, in contrast to results of
the cerulein-treated mice.
Clinically, patients usually experience several episodes of AP before CP is
diagnosed. To make our study clinically relevant, we preestablished CP before WA
treatment was initiated using increased episodes of cerulein each week. We observed
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striking differences in the morphology of the pancreas tissues. The mice receiving WA
showed significantly reduced inflammatory cell infiltration and fibrosis, but mice
receiving cerulein injections had progressed to severe CP, as demonstrated by fibrotic
change. The therapeutic CP model showed an overall increase in Ki67-positive cells
compared with the preventive CP model, albeit the WA-treated group had profoundly reduced damage. We believe that further cerulein-induced damage was reduced after WA treatment, which delayed the advancement of CP. Together, these data support the protective role of WA in the progression of CP.
Recently, ER stress has been recognized as a possible pathological mechanism in
CP progression215. Loss of pancreatic acinar cells by necroptosis due to indefinite
activation of trypsinogen enzymes leads to activation of many inflammatory signaling
pathways234. This loss may also result in ER stress in remaining cells due to the
overwhelming amount of protein synthesis and release requirements. Cerulein has been
shown to activate the ER stress pathway within 30 minutes of injection, although the
stress molecules subside when the mice pancreases recover. Continuous injection of
cerulein results in chronic ER stress that promotes CP215. Confirming this finding, we
also observed an increase in the expression levels of the ER stress markers upon cerulein
injection to induce CP. More importantly, WA was able to efficiently block the
upregulation of all the ER stress markers analyzed. It is still unclear if the mechanisms of
NFκB and ER stress activation are independent of each other in the pathogenesis of CP,
although activation of NFκB and ER stress signaling take place very early upon cerulein
administration215. Interestingly, inhibition of NFκB activation was able to significantly
89
inhibit the ER stress pathway that leads to CP. Figure 4.12 illustrates such of the
signaling pathways operative in CP and the mechanism of blockade by WA.
The role of inflammasome in the development of CP had previously not been
investigated. Inflammasome activation has been identified as a major innate
inflammatory response in several chronic diseases235. Knockdown of key molecules such
as NLRP3, ASC, and caspase 1 reduced the severity of AP236. We have shown for the
first time the role of inflammasome in the pathogenesis of CP and have shown that NFκB
inhibition significantly reduced inflammasome expression and reduced pancreatitis
severity. NFκB is required for the regulation of IL-1β, which gets processed by the
inflammasome complex. It is plausible that due to chronic inflammation, immune cells
infiltrating the pancreas get primed by DAMPs such as HMGB-1, HSPs, or ATP released by damaged acinar cells via TLRs.
This causes assembly and activation of the inflammasome complex that escalates inflammation during CP. Upon cerulein administration, we have observed significant upregulation of HMGB-1, which is a damage marker and initiator of inflammasome signaling. WA profoundly inhibited HMGB-1 expression. NLRP-3, ASC, IL-1β, and IL-
18 levels were also significantly upregulated, suggesting inflammasome activity in cerulein-induced CP. WA was able to strongly inhibit the expression levels of all the above molecules. Therefore, based on these results, it is likely that NLRP3 inflammasome may be partly responsible for enhancing the severity of CP and that WA can be used as a therapeutic intervention to ameliorate this pathway.
90
FIGURE 4.12: Schematic representation of the signaling pathways operative in the incidence of CP and the blockage by WA. During an initial insult to the acinar cells, intraacinar trypsinogen activation leads to ROS release and perturbation in the ER calcium concentrations. ROS can directly activate the kinases responsible for activation of NFκB leading to the production of proinflammatory cytokines/chemokines. Enzymes released from acinar cells cause further necrotic damage to the neighboring cells, while cytokines/chemokines cause local inflammation, recruitment of the innate immune cells. DAMPs initiate inflammasome activation which also requires NFκB activity for maturation. Loss of acinar cells results in UPR in the remaining acinar cells due to protein overload. Chronic ER stress and inflammasome potentiates CP. Withaferin A inhibits activation of NFκB, and prevents inflammatory response.
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CHAPTER FIVE
Summary and Conclusions
Summary
The purpose of this study was to identify inflammatory events that occur in the pancreatic exocrine and endocrine tissue and to use the nuclear factor-kappa B (NFκB) inhibitor withaferin A (WA) to diminish the destruction caused by inflammation.
Inflammation in islets during transplantation is a major issue because it causes significant loss of islets within the first 24 hours after transplantation. The mechanism is termed instant blood-mediated inflammatory reaction (IBMIR). After the islets have been
transplanted, it is very difficult to monitor the number of islets that have been
successfully engrafted and the mass of islets that has been lost in the process. Therefore,
identification and validation of a potential biomarker was necessary to monitor graft loss.
Inflammation in the pancreatic acinar cell leads to acute pancreatitis (AP) and chronic
pancreatitis (CP). We hypothesized that blocking a key mediator of inflammmation,
namely NFκB, using WA could reduce inflammation and the severity of CP.
Therefore, this dissertation addressed three major pitfalls:
1) The significant loss of transplanted islets due to IBMIR
2) The lack of a biomarker to determine islet graft loss after transplantation
3) The unclear understanding of the mechanism of, and cure for, CP
92
Since inflammation is the root cause for these pitfalls, and it is well known that NFκB is a master regulator of inflammatory signaling pathways, I have used WA to block NFκB and overcome inflammatory damage.
Alleviation of Instant Blood-Mediated Inflammatory Reaction in Autologous Conditions through Treatment of Human Islets with NFκB Inhibitors
The successful outcome of islet transplantation has been hindered by several
factors. Inflammation begins when the islets are extracted from their natural environment
and continues for a long time after transplantation. NFκB is a major transcription factor
involved in the regulation of inflammatory signaling pathways177. Therefore, NFκB
blockade using WA and CAY10512 (CAY; a potent analog of resveratrol) was
implemented as a strategy to reduce the activation of IBMIR. A miniaturized in vitro tube
model was developed to investigate the effect of these two compounds on IBMIR174.
Previous studies have reported that isolation and culture of islets leads to production of
proinflammatory cytokines and chemokines that are regulated by NFκB178. Thus, we
hypothesized that once islets are mixed with blood, the innate immune system could be
activated, and this could result in secretion of chemokines by the islets, which recruit
monocytes and neutrophils.
Our data showed that NFκB inhibitors could be potent agents for pretreatment of
islets to ameliorate IBMIR. Both WA and CAY treatment preserved islet viability
compared to the control. Both clinical and in vitro studies have reported immediate islet
destruction after infusion or mixing in autologous blood, shown by elevated C-peptide
and proinsulin levels174. When islets were precultured with WA or CAY, the C-peptide
and proinsulin levels were significantly reduced in the serum, suggesting less islet
93
damage. Contact of islets with blood triggers coagulation, and an immediate increase in
thrombin-antithrombin levels was observed when islets were mixed with autologous
blood. WA or CAY did not affect thrombin-antithrombin levels; however, this was an
expected result because coagulation is an NFκB-independent process.
Islets express high levels of tissue factor (TF), and this interacts with factor VIIa
and activates the extrinsic coagulation cascade. The TF gene is regulated by several transcription factors such as NFκB, Egr, AP-1, and NFAT179. In the case of IBMIR, the
major factor that could induce TF expression in the islets would be the proinflammatory
cytokines. Therefore, blocking TF expression by NFκB would protect islets from IBMIR.
Both WA and CAY significantly reduced TF expression in islets when compared with the control. Since islets were cultured in WA or CAY only for an hour before mixing with autologous blood, a prolonged treatment of NFκB might further reduce TF expression in islets and prevent activation of coagulation.
In this study, the plasma levels of several proinflammatory cytokines were found to be increased after mixing of islets with blood. Proinflammatory cytokines TNFα and
IL-6 and chemokines IL-8, IP-10, MCP-1, and G-CSF increased with time in the control group, and both WA and CAY significantly reduced the level of all the cytokines and chemokines. Infiltration of innate inflammatory cells such as neutrophils into the islets was observed at early stages of transplantation. The islets that were mixed with blood showed an intense neutrophil presence even at 3 hours, whereas the WA- and CAY- treated islets showed significantly reduced infiltration of neutrophils. Future studies using this miniature tube model could analyze the effects of inhibitors for various components of IBMIR, such as different anticoagulants and blockers of the innate immune response.
94
In summary, we have demonstrated that a miniaturized in vitro tube model is an effective
tool to study IBMIR. We have also shown that blocking NFκB is a promising strategy to
alleviate the harmful effects of IBMIR on transplanted islets.
Evaluation of MicroRNA375 as a Novel Biomarker for Graft Damage in Clinical Islet Transplantation
In the present study, microRNA 375 (miR375) has been identified as a biomarker
to quantify human islets and detect damage. In the miniaturized in vitro tube model that
was developed to study IBMIR, a rapid increase in the miR375 levels in the plasma
immediately after mixing islets with blood was observed, which was not seen in islet-
only or blood-only controls. The levels of C-peptide correlated with the levels of miR375. This suggests that the early damage is most likely related to an inflammatory response by the innate immune system. There was a significant increase (~1000-fold) in the serum miR375 level in patients receiving autologous and allogenic islets compared with patients undergoing total pancreatectomy only, suggesting that miR375 is released from islets undergoing damage due to inflammation.
A linear relationship was observed between the amount of miR375 and the islet mass. Using this linearity, it was estimated for the first time that each human islet equivalent consists of about 5.66 × 10-2 fmol of miR375. This information is critical to
quantitatively estimate the number of islets damaged under in vitro and/or in vivo
conditions. This data qualified miR375 as a reliable biomarker to evaluate islet damage,
and the slope of linear regression indicated the amount of miR375 per islet.
Based on miRNA quantification, within 6 hours after mixing with autologous
blood in an in vitro tube model, 40% of islets were damaged. Previously, I showed that
95
the viability of islets was reduced to 56.4% at 6 hours after mixing autologous blood and
islets111. Thus, the amount of damaged islets correlates with the loss of viability data.
Clinical data revealed that 52.4% of transplanted islets were damaged after total
pancreatectomy with islet autotransplantation based on miR375 quantitation, matching
previous observations by other groups. The lack of correlation between miR375 levels 1
hour posttransplant and islet mass could be explained by differences in donor
characteristics and the degree of islet damage during isolation.
Of note, miR375 levels in autologous patients were higher than in allogeneic
subjects, while allogeneic patients showed a higher C-peptide response than autologous
patients. This result is interesting, and the difference in biologic characteristics between
the two biomarkers plays a role in the reverse effect. Elevation of C-peptide levels
represents both the physiologic response of pancreatic β cells to hyperglycemia and
mechanical release due to islet damage198, 199. Autologous recipients undergo major
surgery and are on parenteral nutrition with no metabolic stimulation of transplanted
islets. Also, the islets are isolated from inflamed CP pancreases and hence could be more susceptible to immediate damage than allogenic islets in autologous patients184. Further
investigations on biological conditions that modify circulating miR375 levels are
warranted.
The effects of antiinflammatory agents to improve islet transplantation in patients
were tested. Autologous patients not receiving any antiinflammatory compounds
demonstrated 2 peaks of miRNA elevation, one at 1 hour and the other at 24 hours. The
patients who received antiinflammatory agents such as etanercept or a combination of
etanercept and anakinra had a smaller second peak, suggesting that the islet damage
96
observed after 24 hours may be caused by proinflammatory cytokine–induced apoptosis.
Using the in vitro miniaturized model, it was shown that blocking NFκB using WA
significantly reduced miR375 in the serum compared with control, thus confirming the
role of WA in protection of islets from IBMIR-mediated damage. It is hypothesized that the second peak seen at 24 hours may also be significantly reduced by WA, but an in vitro model is not suitable for the longer time points.
Delineating the Molecular Pathogenesis of Chronic Pancreatitis and Use of NFκB Inhibitor to Prevent Its Progression
CP is an inflammatory disease that causes debilitating abdominal pain, exocrine and endocrine pancreatic insufficiency, and severely compromised quality of life. To date, there is no curative treatment for CP or a unified approach to control its progression.
Cerulein-induced pancreatitis is widely accepted as a clinically relevant model for recurrent acute necrotizing idiopathic pancreatitis. The role of NFκB in the development of CP has been widely documented. Therefore, it was hypothesized that inhibition of
NFκB using WA would protect the mice from progressing to CP.
Using a cerulein-induced AP model, it was observed that WA reduced disease severity and inflammation. The ability of WA to protect mice from recurrent episodes of
AP that eventually lead to CP was tested. WA-treated mice pancreases demonstrated reduced inflammation based on histopathological analysis compared with pancreases of mice receiving cerulein injection alone. Serum amylase levels, immune cell infiltration,
Ki-67 staining, and inflammatory cytokine levels in the pancreas were lower in the WA treatment group than in the cerulein-treated mice.
97 To make the experiment clinically relevant, CP was induced prior to WA
treatment. The morphology of the pancreas tissue in WA-treated mice had significantly less damage than that of the non-WA–treated mice. The mice receiving WA showed significantly reduced inflammatory cell infiltration and fibrosis compared to the cerulein-
only–treated mice. These data suggest that further cerulein-induced inflammatory damage was reduced after WA treatment, which delayed the advancement of CP.
CP may result in endoplasmic reticulum (ER) stress in remaining cells due to the overwhelming amount of protein synthesis and release requirements. An increase in the expression levels of ER stress markers was observed in CP mice upon cerulein injection.
WA was able to downregulate the expression of all ER stress markers analyzed.
Interestingly, inhibition of NFκB activation resulted in significant inhibition of the ER stress pathway that promotes the severity of CP.
The role of inflammasome in the development of CP had not been previously investigated. With the current study, the emergence of inflammasome in the pathogenesis of CP has been identified for the first time, and WA significantly reduced inflammasome expression and reduced pancreatitis severity. Cerulein administration resulted in significant upregulation of high-mobility group box-1 (HMGB-1), which is a damage- associated molecular pattern molecule and initiator of inflammasome signaling. WA profoundly inhibited HMGB-1 expression. WA was able to strongly inhibit the expression levels of inflammasome molecules that were upregulated by cerulein.
Therefore, based on these results, it is likely that NLRP3 inflammasome may be partly responsible for enhancing the severity of CP and that WA could be a therapeutic intervention to ameliorate CP.
98
Conclusion
In conclusion, the role of NFκB in causing inflammation to the exocrine (acinar) and endocrine (islet) compartments of the pancreas has been identified. I have demonstrated that WA, a potential inhibitor of NFκB, can resolve inflammation in the islets and acinar cells. WA can be used as a potential therapeutic treatment to improve islet transplant outcomes and also to ameliorate CP. Finally, I have also identified miR375 as a potential biomarker for islet damage during islet transplantation.
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