Coeliac Disease

Coeliac Disease

• Is a Gluten Sensitive Enteropathy (GSE) • It is a relatively common disease that affects the small intestine which

involves an immune response to dietary gluten, but only in genetically predisposed individuals

• Arises from the word: Koiliakos = Greek for belly • Also known as Gee-‐Herter Disease as it was first described in the modern era

by Samuel Gee (1888) and later by Christian Herter (1908) • Prevalence of 1:100 -‐ 1:300 -‐> relatively common especially in caucasian

populations located in Europe, N. America, Australia, South Africa and hispanic populations of S. america. It is also present in the Middle East. Highest prevalence noted in the Sahawari people of Sub-‐saharan Africa ◦ It is not seen in African and Oriental populations

Origins

• Cereals have only been cultivated over the last 20,000 years -‐> so it has only been a short period of time that our immune systems have been coexisting with gluten -‐> so the history of coeliac disease is relatively short

• You can trace the spread of Coeliac disease from the fertile crescent located in the Nile Delta -‐> then there was a migration northwards after the end of the first ice age

Histopathology

• Immune activation in the gut mucosa • Characterised as having:

◦ Increased Intra-‐epithelial lymphocytes (IELs) ◦ Increased lamina propria lymphocytes (LPLs) ◦ Crypt Hyperplasia -‐ the crypts take over the villi ◦ Villous Atrophy -‐ loss of villous height

Grading of Severity (Marsh, 1992)

• Grade 1: Presence of Increased Intra-‐epithelial lymphocytes (Normally there is up to 25 lymphocytes/100 enterocytes in mucosa -‐ roughly 60% of T Cells in body are present here)

• Grade 2: IELs, Crypt Hyperplasia, LPLs • Grade 3: Reduction in Villous height • Grade 4: Flat , atrophic villi

Anatomical Site at which Coeliac Disease targets

• Coeliac Disease results in loss of surface area for absorption in the proximal small intestine

• Causes malabsorption of: ◦ Fe ◦ Folate ◦ Fat ◦ B12 -‐ to a lesser extent

Presentation

• It was thought in the past, that Coeliac disease was a disease of childhood • But, we now know that it can occur at any age

◦ But is the coeliac disease diagnosed at old age, a disease that has not been detected for a long time or has just been triggered

• It was also thought that all patients would suffer weight loss and diarrhoea -‐ however, we are now seeing a much wider range of clinical presentations (even in obese individuals)

• Normal clinical signs: ◦ Diarrhoea ◦ Weight loss ◦ Fatigue ◦ Anaemia ◦ Abdominal pain (often misdiagnosed as irritable bowel syndrome) ◦ Loss of bone density -‐> can result in osteoporosis in later age

• However, a large number of patients (6/7ths) are completely asymptomatic or have mild, chronic, insidious symptoms

The Environmental Trigger

• It was only in 1950, that it was discovered that Coeliac disease was due to some sort of a toxic factor in cereals (WK Dicke, 1950) ◦ Discovered, in the Dutch Winter famine in 1944, when children with

Coeliac disease gained weight when everyone else in the population lost weight

◦ Subsequently, the children with CD, lost weight when everyone else started putting on weight, following the famine

• Gluten is found in Wheat, Rye and Barley ◦ Copy diagram slide 12

• Nature of Gluten: ◦ It is not a single protein rather an insoluble proteinacious fraction

(75-‐85%) present in Wheat dough ▪ It can be further solubilsed in ethanol, giving rise to a Gliadin

Component (Prolamins) ▪ 35% is glutamine (hydrophilic) , 15% is proline

(hydrophobic), 19% is hydrophobic amino acids ▪ Has few charged side groups ▪ And is resistant to proteolysis largely due to the proline

in it ▪ It is found in the wheat endosperm and it has not 3D

component/constraints to it -‐> It is there to store all things needed for the seed to germinate

▪ It also maximises the Nitrogen in the protein (f) the glutamine residues (which has an extra amino group on the side) which is good for the germinating seed

▪ The insoluble fraction (70%) is Glutenin (can be HMW or LMW)

▪ Copy slide 13 ◦ The glutenins and gliadins contain the toxic components of gluten,

particularly the gliadins ◦ The Wheat Karyotype is Hexaploid ◦ Wheat has been bred to maximise the gluten content

▪ Makes wheat sticky Testing for toxicity

• Organ Culture ◦ Take biopsies of the organ and incubate it with particular peptides of

the gluten -‐> this can replicate the whole immune response in vitro • In vivo testing

◦ Take a coeliac patient who has just recovered and test the patient with different proteins and peptide sequences

• In vitro lymphocytes stimulation tests ◦ Proliferation of lymphocytes with different peptide sequences can be

assessed • Copy slide 15

Testing for toxicity has revealed certain peptides that are toxic and not toxic in gliadin

• There are a particular group of peptides that are toxic, but much more toxic when deamidated

• Within the gliadin sequence, there is a 33 amino acid oligopeptide (57-‐89) which contains the 3 main immunodominant epitopes which cause the problem in CD (Shan et al, 2002)

• Since, this is very difficult to digest, due to the proline residues, we actually see this 33-‐mer-‐peptide in physiological digestion conditions of gliadin.

• So we are exposed to these toxic components of Gluten normally in the gut It is a T-‐Cell Mediated Condition -‐ Evidence:

• T Cell stimulation by gliadin peptides has been demonstrated in vitro • Gluten challenge leads to proliferation of CD4+ CD25+ Treg Cells

in the Lamina Propria ◦ It is an activation marker

• Increased TNFalpha, IL2, IL12 and IFNg has been detected after gluten ingestion

• Experimental models: ◦ IL12/TNFa injection – mimicking organ damage described earlier in

vitro ◦ GVHD similarity – which is a lymphocyte mediated response that is

removed by Cyclosporin (a T Cell inhibitor) ◦ T-‐cell mitogen/anti-‐CD3 activation in fetal gut explants ◦ TCR transgenic mice

• HLA Restriction ◦ Those patients with coeliac have a particular type of HLA ◦ This is the most obvious sign, that this is T Cell mediated

Genetic Factors

• 8-‐14% of first degree relatives with condition (Carter et al, 1959) ◦ Relatively high proportion

• 75% concordance of identical twins (Greco,2002) • 17% concordance of haploidentical siblings (siblings with same HLA)

◦ So not all due to the HLA subtype, so there are clearly other genetic factors

• >95 % of CD patients have HLA DQ2 or DQ8 ◦ 28%/20% controls respectively (Schulz et al, 1981) ◦ Particular alleles of DQ2 or DQ8 are responsible (Arentz-‐Hansen

2002) ◦ Copy slide 20

So, it is likely the toxic antigen (the gliadin peptides which are processed by APCs) is being presented to CD4 T Cells by HLA DQ2 or 8 by antigen presenting cells The Negative Charge Problem

• We know the epitopes in gliadin that cause the toxicity and the binding groove within the HLA DQ molecule -‐ but there needs to be a negative charge to bind to this groove. However, the gliadins contain a lot of glutamine which carry a positive charges

• How is this problem solved? B Cells in Coeliac Disease

• We know that B Cells can make autoantibodies in CD such as: ◦ Anti-‐reticulin Ab ◦ Anti-‐endomysial Ab (AEB)

▪ These are a very good indicator of CD (but not perfect!) ▪ Produced by intestinal B Cells ▪ Stimulated by gliadin in vitro

◦ Anti-‐gliadin Ab • In 1997, Dietrich et al found that the antigen recognised by AEB was actually

Tissue Transglutaminase (TTG) ◦ It is a very large enzyme, involved in cross-‐linking of connective

tissue proteins and in healing ◦ Generated following NF-‐kB binding to promoter ◦ Much more found intracellularly than extracellularly ◦ Contains 5 members ◦ It also has the ability to deamidate glutamine residues in gliadin

• Solving the Negative Charge Problem ◦ TTG can deamidate the glutamine residues in Gliadin -‐> lose positive

charge -‐> becomes -‐vely charged -‐> The gliadin epitope can now bind to the binding groove within HLA DQ and get presented to T Cells in the

• • Why are these produced in Coeliac disease?

• Probably due to non-‐professional APCs -‐ B Cells ◦ The BCR Ig (Anti-‐TTG) can recognise the gluten-‐TTG complex and

endocytose it. Then it can present processed TTG to CD4 T helper Cells via MHC Class II -‐> these can then result in Anti-‐TTG antibodies

◦ 15% are TTG Ab negative , but still have an identical phenotype of disease

◦ So it is believed that these Abs don't really contribute anything significant to the pathogenesis of the disease

◦ However, Dermaititis Herpetiformis is believed to be caused by these Abs?

◦ Others Abs may also be involved in the pathogenesis of CD -‐ IgA lining the intestinal mucosa can take up luminal human peptides (like gliadin) which are then transported by CD71 across the mucosal cells of the gut, and then secreted across the basolateral membrane -‐> TTG can then modify the gliadin and these IgA/gliadin complexes can activate APCs which present the gliadin to CD4 T Cells

◦ Copy diagram slide 31 Stromal Cell Involvement

• Inflammatory cytokines produced by the CD4 T helper cells can such as TNFalpha and IL-‐1B can induce metalloproteinase expression in mesenchymal cells isolated from fetal intestine

• Exposure of fetal intestinal explants to pokeweed mitogen causes architectural damage similar to CD and elevated NNP levels

• Similar changes seen with addition of Stromelysin 1 (MMP3) to fetal intestinal explants, and are inhibited by anti-‐stormelysin Abs

• Mesenchymal cells (like fibroblasts?) and TCRgammadelta IELs produce Keratinocyte Growth Factor -‐> maybe responsible for crypt hyperplasia

Copy summary diagram slide 33 and notes Why is there a genetic predisposition in some people?

• A transgenic mice has been produced that has got a Human HLADQ8 and Human CD4 T Cells injected into them

• But when they are exposed to Gluten, these mice don't get an active inflammatory response. Instead, you get expression of regulatory cytokines like TGFB, and IL10

• So, something else must be occurring in humans that is stimulating or triggering an active inflammatory response -‐ possible causes: ◦ Infection

▪ Many children get a rotavirus infection before presenting with CD

▪ With the introduction of the rotavirus vaccine, could there be less CD incidence in children?

◦ The Adenovirus Hypothesis ▪ The alpha-‐gliadin sequence shows homology with the

Adenovirus 12 E1b protein

▪ It was proposed in 1984, by Kagnoff et al, that there could be cross reacitivyt of Ab 12 E1b protein Abs with Gliadin ▪ Evidence for: CD patients have a higher titre of Ad12

Abs than controls ▪ Evidence against: Cross reactivity of Abs not shown in

patients No Adenovirus in the mucosa of CD patients The Homologous Gliadin peptide is only weakly immunogenic

▪ So, overall, there is still a small possibility it could be a trigger, but unlikely to cause the condition.

◦ Pro-‐Inflammatory Cytokines ▪ IFNs are used for treatment in a variety of diseases

◦ Gluten Dose Threshold Effect ▪ Some patients are more susceptible to gluten than others ▪ They might have the genetic susceptibility, but only present

once the gluten dose crosses their threshold ◦ Inflammatory threshold Effect

▪ ??? ▪ Key cytokines involved in CD: IFNalpha, IL15, IL21 ▪ Copy diagram slide 36

• Recent Advances in CD Genetics: ◦ Candidate Gene approach -‐> found MYO1XB, and CTLA-‐4 being linked

to CD ◦ GWAS studies have identified new potential gene targets (e.g. Hunt et

al, 2008) ▪ 7 targets including IL18RAP, IL2-‐IL21, CCR3, SH2B3 ▪ Some, overlap with Type 1 Diabetes and ??Ulcerative Colitis??

◦ However, these studies have only contributed 3-‐4% of the heritability ◦ HLA association contributes to about 40% of the heritability of CD! ◦ 2nd generation genes identified by GWAS have identified more

polymorphisms that may include genes involved in T Cell development, Innate Viral Replication, T & B Cell co-‐stimulation, Inflammatory Cytokines

Innate Immune Response in CD

• IL 15 ◦ There probably needs to be a danger signal to trigger

an active immune response ◦ Maiuri et al, 2003 proposed that IL15 is the epithelial danger signal ◦ It has been shown that its production is induced by enterocytes after

incubation with Gluten ◦ Different part of the gliadin sequence (compared to the epitopes

before) is responsible for increased IL15 production ◦ IL15 is responsible for:

▪ Increased T Cell stimulation ▪ Increased Enterocyte Apoptosis ▪ Potentiation of adaptive immune response

▪ Induction of heat shock proteins and stress proteins such as MICA on the surface of enterocytes (Hue et al, 2004) ▪ These proteins are also the targets for NKC mediated

cell lysis ◦ So, now we are looking at immune responses in the epithelia for the

innate immune response in CD (not just the Lamina propria for antigen presentation and the adaptive immune response)

Intraepithelial lymphocytes

• Diverse Phenotypes • 98% CD3+, CD8+ • 2 types of TCR:

◦ Alphabeta ◦ Gammadelta

• Most of them are T Cells ◦ 60% of all T Cells in the body are found in the

• Many of them are also NKCs -‐ they can carry TCRs as well • They may provide a bridge between innate and adaptive immunity

Zonulin

• Is an endogenous modulator of epithelial tight-‐junctions • Zonulin is produced by enterocytes and it increases permeability of

epithelial barrier • Gliadin sequence 131-‐150 can be recognised by a chemokine receptor and a

MyD88-‐dependent pathway stimulate the production of zonulin in enterocytes -‐> increase permeability

The big picture -‐ gliadin can affect the gut mucosa in 3 ways

• A non-‐immunoglobulin epitope of gliadin (31-‐47) leads to stimulation of IL15 production from enterocytes-‐> stimulates the proliferation of IELs and their activation and expression of NKC effector molecules. It also stimulates the production of stress molecules on enterocytes -‐> activation of NK properties of IELs (the NKG2D cells)-‐> Secretion of perforin/granzymes -‐> damages the epithelial cells -‐> increased permeability of epithelium -‐> allows 33mer (57-‐89) and 17 mer toxic gliadin peptides to enter submucosa and activate the adaptive immune responses

• The 131-‐150 sequence of gliadin can also cause increase in zonulin and thus epithelial permeability

• Copy slide 44, 45 Malignancy in Coeliac Disease

• There is an increase risk of cancer in CD (not significant in those with a gluten-‐free diet) esp. small intestine

• Copy slide 46 • Risk of breast cancer is reduced in CD • There is one type of cancer, that is almost completely unique to CD:

Enteropathy Associated T Cell Lymphoma (EATL)

◦ 40-‐100 fold increase in CD (but still pretty rare -‐ normal incidence is just 1/1000000??)

◦ 31-‐69 fold increased risk of dying from lymphoma ◦ EATL is associated with a lot of the deaths in CD -‐ However, this

is decreasing? ◦ But it has a poor prognosis -‐ 5 year survival rate is around 25%

▪ Copy slide 48 ◦ Why does it happen?

▪ It is due to the ongoing stimulation of intestinal lymphocytes (the T Cells) in CD

• Refractory Coeliac Sprue or CD ◦ CD unresponsive to Gluten Restriction ◦ Causes of lack of symptomatic improvement include:

▪ Dietary non-‐compliance ▪ Non-‐coeliac sprue ▪ Lymphocytic colitis (associated with CD) ▪ Lactose intolerance ▪ Coincidental conditions ▪ They carry atypical IELs (non-‐CD3, non-‐CD8 lymphocytes)

▪ They have rearranged their T Cells and do express intracellular CD3

▪ There is a clonal proliferation of these T Cells -‐> progression to lymphoma after 3 years, in about 50% of the cases

▪ Thus, refractory sprue is a half way stage between CD and lymphoma

▪ The cellular phenotype seen in Refractory sprue is identical to EATL??

◦ Mechanism of RCS/EATL: ▪ There appears to be some sort of autonomous replication,

maybe (f) chromosomal damage, in the lymphocytes as a result of the ongoing proliferation -‐> refractory CD -‐> Malignant proliferation ▪ Particularly likely in patients who have had undetected

mild CD for a long time, and can present in old age ▪ Copy slide 54

Novel Treatements in CD

• The only current treatment is getting rid of gluten out of the diet ◦ However, this can be difficult to get out of the diet, because it is

present widely in the diet. • The other option is to decrease the immune response e.g. by

calcinuerin inhibitors. However, this can cause a lot of unwanted side effects, so it is preferable to try dietary methods first.

• Copy slide 56, 57

Coeliac Disease

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