What is the epidemiology of IBD?
- Chronic inflammation of the intestine.
- Crohns → can affect any part
- Ulcerative colitis → affects the colon
- Prevalance → 2.5 million in Europe, 1.3 million in USA
- Age of onset → Crohns 20-30, Ulcerative colitis 30-40
- Not diseases of frailty – often affect young, healthy adults.
What are the general causes of IBD?
- Breakdown in intestinal immune homeostasis, leading to inflammation
- Due to barrier defects, or immune regulation effects (enhanced T cell responses against the microbiota)
- Macrophages and DCs reported to secrete more IL12 → induces a Th1 response → TNFa and IFNy
- They can also produce more IL-6, IL-1B, IL-23 and TGFB → induces a Th17 response.
What causes the immune breakdown?
Environmental factors (not well understood, but some factors thought to be involved are):
• Diet
• Smoking → potentially protective!
• Use of antibiotics
• Infection
• Stress
→ Even though not well understood, is still important because only 35% concordance in identical twins
Genetic risk factors:
• GWAS have screened for genetic differences in disease-affected individuals
• International IBD genetic consortium, 2010 studied >75,000 IBD patients and healthy controls. 161 IBD susceptibility loci identified
• Most of these associated with the immune system, eg)
− NOD2 (involved in recognition of microbial antigen)
− STAT3 (involved in Th17 biology)
− IL-10 associated genes
− TGFB associated genes
What are the current treatments for IBD?
- Steroids to dampen the immune response. If unresponsive…
- Infliximab (blocks TNFa). If unresponsive…
- Surgery to remove the affected area of the intestine.
• In order to develop better targets for IBD therapy, we need better cellular and molecular understanding of the disease.
Why is mucosal immune system necessary?
- The thin layer of mucosal epithelium is the only physical barrier against invasion of the underlying tissues by pathogens and the body’s own commensals.
- These surfaces therefore require continual protection against invaders
- Mucosal surfaces (upper & lower respiratory tract, urogenital tract, GI tract) represent an enormous area to be protected – the small intestine has a surface area of 400m2, almost 200times that of the skin
- Due to their function in absorption, respiration, reproduction etc… the mucosal epithelium is thin and permeable, and can be breached easily. The importance of these tissues to life means that their barrier function needs to be supplemented by defenses provided by the mucosal immune system.
- Mucosal surfaces are portals of entry for the majority of pathogens, but also a vast array of foreign antigen that isn’t pathogenic → 30-35kg food antigen per year, and 1013 commensal organisms per gram of content.
- Generating protective immunity against these harmless antigens would be inappropriate an wasteful, with immune responses of this kind known to b the causes of Celiac disease and IBD.
What are the distinctive features of the mucosal immune system?
• From the traditional view of immunology, the mucosal immune system has been considered to be a minor sub-compartment of the immune system
• In terms of size and function, this is inaccurate → forms the largest part of the body’s immune system, containing 75% of all lymphocytes and producing the majority of Ig.
• Has a number of distinct features when compared with the lymph nodes and spleen (systemic immune system):
− intimate interactions between mucosal epithelia and lymphoid tissue
− Discrete compartments of diffuse lymphoid tissue and more organized structures such as Peyer’s patches, isolated lymphoid follicles and tonsils
− specalised antigen uptake mechanisms, eg, M cells
− Activated effector T cells predominate even in the absence of infection
− Multiple activated regulatory T cells present
− Secretory IgA
− Presence of distinctive microbiota
− Active downregulation of immune responses
− Inflammatory macrophages and tolerance-inducing DCs.
• May have been the first part of the vertebrate adaptive immune system to evolve → co-evolved with the need to deal with the commensal bacteria that coevolved with vertebrates.
Outline the structure of the intestine
- Small and large intestine lined by simple columnar epithelium and overlying mucous layer, upon and within witch commensals reside
- Organisation not homogenous across both compartments → small intestine has villi for increased surface area for food absorption, so mucosal layer discontinuous and vili tips often not covered (Johansson et al, 2011). Colon functions to absorb water so is flat with thick, disorganized outer mucous layer and stratified inner layer firmly adhered to the epithelium (Atuma et al, 2001).
- Microbiota density also changes – 103 in small intestine and 1012 in colon.
- In the steady state, commensals in colon only found in outer mucous layer, but colonization of the inner layer can be seen in cases of IBD
Outline the 3 main roles of the microbiota in human health.
- Food digestion (energy/nutrient release) → human genome encodes fewer than 20 enzymes for sigestion of complex carbs. Instead, many are broken down by the gut microbiota and then absorbed by the gut. Depending upon the specific composition of the gut microbiota, metabolism of other substances, including drugs, can also be altered in some individuals. Humans are unable to synthesize most of the vitamins required to support health. Whilst we derive many of these essential nutrients from food sources, the gut microbiota is able to produce vitamin K and most B vitamins including folates, thiamine, riboflavin and biotin. Uptake of microbially produced vitamins occurs predominantly in the colon.
- Protection from intestinal infection (niche against pathogens)
- Regulation of intestinal immune system (project!)
Describe the components of the GALT.
• Peyer’s patches:
− Important sites of initiation of immune responses
− Domelike aggregates of lymphoid cells that project into the lumen
− 100-200 in the small intestine
− Much richer in B cells than the systemic lymphoid organs
− Sup-epithelial dome region is rich in DCs, T cells and B cells
− The follicle-associated epithelium separates the lymphoid tissue from the gut lumen – contains the specialised M cells.
− M cells don’t secrete enzymes or mucous and lack a thick surface glycocalyx, so they are in direct contact with the gut lumen
• Isolated lymphoid follicles:
− Several thousand throughout small and large intestine
− Composed of an epithelium containing M cells overylying organised lymphoid tissue
− Contain mainly B cells
− Develop only following gut colonisation by commensals.
− Arise from small aggregates in the intestinal wall called cryptopatches
• MLNs
− PPs and ILFs connected by the lymphatics to the MLNs
− Largest lymph nodes in the body
− Small intestine and colon drain to distinct nodes within the MLN chain.
Describe the distribution of immune cells within the intestine.
- Epithelium predominantly CD8+
- Lamina propria CD4+, CD8+, DCs and B cells.
- 10-15 lymphocytes for every 100 epithelial cells – makes the IELs one of the largest populations of lymphocytes in the body.
Where is there a need for such tight immune regulation within the intestine?
- IBD thought to be driven by effector T cells, but a common feature in the intestine is that even in the absence of disease, most of the lymphocytes have the appearance of activated effector cells that have seen antigen and received necessary costimulation.
- Healthy intestinal mucosa therefore displays many features of a chronic inflammatory situation → mucosa and barrier therefore represents a significant barrier in the protection against pathogens and prevetion of unwarranted immune responses against the microbiota.
- Barrier not only physical, but also secretes anti-microbial peptides and is responsible for the transcytosis of IgA.
- Importance of these barriers highlighted by the onset of IBD in mice with compromised epithelial layer integrity or defective mucous production.
- However, T cells have an activated phenotype, so even with the presence of barriers, other immune regulatory mechanisms needed to prevent overactive effector responses to food antigen or commensals that may penetrate.
What is the role of IgA in intestinal immunity?
− Binds to the layer of mucous coating the epithelial surface via carbohydrate determinants in the secretory component.
− Involved in preventing invasion by pathogens and also maintain balance between the host and the commensals
− IgA inhibits microbial adherence to the epithelium, assisted by the wide and flexible angle between the Fab pieces of both IgA isotypes, allowing efficient bivalent binding to large antigens such as bacteria.
− Secretory IgA can also neutralise microbial toxins and enzymes both in the lumen and inside epithelial cells
− The resulting IgA:antigen complexes are rexported to the gut lumen from where they are excreted from the body.
Describe the structure of TGFB.
- One of over 40 members of a superfamily of cytokines
- Exists as 3 highly homologous isoforms with distinct spatial and temporal expressions. TGFB1 important in immunity
- Synthesised as latent pro-peptides → 75kDa LAP and 25kDa mature cytokine associated to form the small latent complex (Munger et al, 1997).
- LAP linked by disulphide bonds to another family of proteins known as latent TGFB binding proteins (LTBPs), forming the LLC which is targeted for secretion into the ECM.
- LAP acts as a molecular straightjacket – encapsulating the mature TGFB and preventing it binding to the receptor.
- LTPB reinforces the straightjacket and creates a reservoir of stored latent TGFB in the ECM awaiting activation.
What is the general role of TGFB?
- Highly pleotropic cytokine, receptors expressed on virtually all cell types.
- Controls cellular proliferation and differentiation throughout the body
- Clinical significance in cancer, kidney disease, atherosclerosis and asthma.
- Key role in regulating the immune system → mice lacking the TGFB-1 isoform die early of multi-organ failure characterised by tissue necrosis and multifocal inflammation (Kulkarni et al, 1993).
How does TGFb regulate: CD4+ T helper cells Tregs Th17 cells Th9 cells CD8+ T cells DCs?
CD4+ T cells:
• Letterio and colleagues found that the inflammation seen in TGF-B KO is revered when the anti-CD4 antobodies are administered – suggests a key function is the regulation of effector CD4+ T cell responses.
• Inflammatory response resulting from expression of the dominant negative TGFB-RII associated with enhanced naïve T cell differentation into Th1 or Th2 effector cells.
• TGFb suppresses differentiation of Th1 and Th2 by downregulating their respective cell-specific TFs, T-bet and GATA-3 (Gorelik et al).
• Inhibits cytokine production by fully differentiated Th1 cells, but little effect on fully differentiated Th2
Tregs:
• Knockout of Foxp3 causes autoimmunity similar to that seen in TGFb knockout (Fontenot et al, 2005)
• Role in generation of iTRegs well documented → in the presence of IL2, TGFB converts naïve CD4+ T cells to iTregs by inducing de novo Foxp3 expression.
• TGFB knockouts therefore have reduced Foxp3 Treg population in the peripheral pool
• This induction is particularly common in the intestine
• Studies also shown that it is involved in development of nTRegs, with deletion of TGF-BR preventing nTReg production in neonatal mice.
Th17:
• In the presence of IL1, IL-6 and IL-21, TGF-B stimulates proliferation of proinflammatory Th17 cells by promoting expression of RORyt.
• Mice with reduced Th17 cells as a result of non-functional TGF-BR protected from the inflammation associated with autoimmune encephalitis.
Th9:
• In combination with IL-6, upregulates PU.1, the master transcription factor for Th9 cells
• Associated with allergic responses and associated tissue inflammation
CD8:
• In the presence of TGFB, CD8+ T cells fail to gain their full effector function through inhibition of cytolytic proteins and disruption of the death receptor cytotoxic pathway.
• Also suppresses cytokine production by fully differentiated CD8 cells
• However, shown to favour expansion of a specialised subset of CD8aa+ T cells in the intestine, which have a regulatory role in mucosal immunity.
DCs:
• Mice with deletion of the TGFB-RII produce more IFNy, which reduces their ability to induce Tregs, and the mice die early on from multi-focal inflammation
How is TGFB activated?
- Given its diverse functions, its activity has to be tightly controlled
- No evidence to date that cells may secrete TGF-B in an active form, un-asscociated with the LAP. Additionally, it appears that TGF-B is synthesized in excess (Annes et al, 2003). → Therefore, activation likely to be the major regulatory step.
- Heat, extreme pH, ionizing radiation all shown to activate TGF-b in vitro
- Recent evidence suggests fundamental activators of TGF-B in vivo are members of the av family of integrins.
Integrins:
• Transmembrane cell-adhesion receptors involved in linking cells to the ECM
• Heterodimers, capable of mediating bi-directional signaling
• Composed of alpha and beta subunit, 18 alpha and 8 beta that can combine to make 24 integrins
• 6/24 shown to bind TGF-B via an argigine-glycine-aspartate (RGD) sequence present in the LAP.
• Integrin involvement first suggested by Munger and colleagues → created knock in mice with a point mutation that converted RGD to RGE, preventing integrin binding to TGFB – these mice had similar phenotype to TGFB1 knockout.
• Further work in this lab demonstrate role for avb6 and avb8 integrins specifically → integrin B8 knockout mice treated with an anti-avb6 antibody (giving dual avb6 and avb8 ablation) found to present with almost the same findings as that seen in TGFB1 knockouts.
Mechanism of integrin mediated activation:
avb6:
• A contractile force mediated through interactions with the B6 domain with the actin cytoskeleton cause a conformational change in the latent TGF-B complex
• This results in an unfastening of the straight jacket and subsequent exposure of the binding sites
avb8:
• The cytoplasmic domain of the B8 integrin does not bind the cytoskeleton, therefore activation must be by another method.
• Inhibition of MMP-14 was found to inhibit TGFB activation, and this protease co-localises with avb8 on the surface of cells
• Therefore, a model was proposed in which the integrin acts as a form of scaffold, binding to the LAP nad present latent TGF-B to MMP-14 for activation. Indeed, MMP-14 has been shown to cleave the SLC at a protease sensitive site near the LAP amino terminus (Mu et al, 2002) and can also release the LLC from the ECM via cleavage of the LTBP.
• Proteolytic cleavage of the LAP allows the active TGF-B cytokine to difuse away from the site of initial activation and act on cells further afield (although not known to what extent it does diffuse way). This is in contrast to avb6 mediated activation, whereby ECM tethering by the LTPB gives spatial regulation at the cell surface.
- avb9 is more widely expressed, with avb6 mostly restricted to epithelial cells
- Travis and colleagues have shown avb8 to be particularly expressed in cells of the immune system, namely the CD4+ T cells and DCs (Travis et al, 2007)
- Mice lacking this integrin in all cells die before birth of brain haemorrhage (this ingegrin has an important role in neurovascular development)
- Deletion of avb8 specifically on leukocytes results in autoimmunity characterized by enhanced T cell activation, suggesting leukocyte expressed avb8 has a non-redundant role in inflammation and T cell homostasis (Travis et al, 2007).
Describe the properties of PP and LP DCs.
DCs:
• During health → in the presence of commensal bacteria, production of PGE, TGFB and TSLP inhibits DC maturation
• During infection → invasive microorganisms penetrate the epithelium to activate DCs
But it is not as simple as this
• Difference subsets of DCs in the intestine have different functions, and these can be anti- as well as pro-inflammatory.
• PP DCs are mostly CD11b+ and express CCL20 produced by follicle-associated epithelial cells
− In resting conditions, these DCs remain beneath the epithelium and produce IL-10 in response to antigen acquired from M cells → prevents priming of T cells to become effector cells.
− During infection, these DCs are rapidly recruited into the epithelial layer of the PP in response to CCL20 released by epithelial cells in the presence of bacteria. Bacterial produces also stimulate DCs to express co-stimulatory molecules to prime effector T cells.
• LP DCs comprise a unique subset that make important contributions to maintaining tolerance, particularly against food antigen.
− Most LP DCs express CD103 (integrin aeb7)
− Once loaded with antigen, CD103+ DCs migrate to T cell areas of the MLNs
− In the MLNs, they interact with naïve T cells, and their unusual property of retinoic acid production allows them to induce gut homing receptors a4b7 and CCR9, enabling the T cells to efficiently return to the intestinal wall as differentiated effector T cells.
− LP DCs response poorly to inflammatory stimuli such as microbial TLR ligands, so when they arrive in the MLN under resting conditions, they promote the generation of iTRegs. This process is also retinoic acid dependent and assisted by TGF-B.
− The anti-inflammatory properties of CD103+ DCs is actively promoted by factors constitutively produced in the mucosal environment → TSLP, TGF-B, PGE2. Additonally, IL-10 from macrophages in the mucosa retains DCs in a quiescent state and maintains the local TReg population.
Describe the initial experiments indicating avb8 on DCs is important for intestinal immune homeostasis.
- When avb8 is knocked out in DCs or T cells, the mice didn’t die of multi-organ inflammation at 3 weeks → so this integrin isn’t the full answer for TGF-B activation.
- However, mice lacking avb8 specifically on DCs develop the same autoimmune colitis phenotype as mice lacking avb8 on all leukocytes, and is characterised by a reduced population of TRegs and consequently increased effector T cell activation in the intestine (Travis et al, 2007) → therefore, integrin avb8 on DCs is important in maintaining intestinal immune homeostasis.
- This lack of avb8 does not affect the ability of DCs to mature and gain their effector function, however it was associated with decreased TGF-B activity → highly suggests that activation of TGF-B by avb8 on DCs is crucial for inducing TRegs in the intestine.
What are the two potential explanations for why the pathology associated with avb8 KO on DCs is restricted to the gut.
- Explanation 1 → because the intestine is a site of such high immune load, it is only really an important anti-inflammatory pathway within the intestine. Indeed, deletion of this integrin in GF mice doesn’t result in colitis.
- Explanation 2 → The CD103+ DCs are specialized for inducing TRegs. Because TRegs need TGF-B, and CD103+ DCs induce TRegs, is there a link? Is it that these CD103+ DCs are specialized to activate latent TGFB via avb8?
Describe the experiments that showed why the pathology of avb8 KO DCs is restricted to the gut.
Worthington et al, 2011:
• Was shown that CD103+ DCs expressed vastly higher levels of avb8 than CD103- DCs
Is this functionally important in activating TGFB?
• Using a TMLC assay (in which cells express a luciferase construct under the control of a TGF-B responsive element), it was shown that CD103+ DCs have enhanced ability to activate TGF-B. Lack of integrin avb8 on gut CD103+ DCs abolishes this ability.
Is this functionally important in activating TRegs?
• CD4+ T cells from GFP-Foxp3 mice were sorted to high purity by flow cytometry.
• These cells were then cultered with DCs, and they looked to see how many of them became green – and hence are expressing Foxp3.
• It was shown that CD103+ DCs have enhanced ability to activate TRegs. Lack of integrin avb8 on gut CD103+ DCs abolishes this ability.
• Reduced TReg induction by CD103+ DCs lacking avb8 is rescued by addition of active TGF-B.
Is this important in vivo?
• Travis et al, 2007 demonstrated a reduction in colonic TRegs in mice lacking avb8 expression on DCs.
Current model:
• CD103+ DC express high levels of avb8
• This enables them to activate high levels of TGF-B, which is known to be important in driving high levels of TRegs.
• Important in suppressing self-harmful T cells, because if we disrupt this pathway, we get autoimmune colitis.
• Retinoic acid can only induce TRegs if you have avb8-mediated TGF-B activation
Discuss the role of avb8 on DCs in activating Th17 cells.
Melton et al, 2010:
• Showed that integrin avb8-DC-TGFB pathway is also important in the induction of Th17 cells as well as TRegs.
• Th17 cells can promote a variety of autoimmune diseases, including psoriasis, multiple sclerosis, rheumatoid arthritis and IBD
− Th17 cells nearly absent in the colons of mice lacking avb8 expression on DCs
− DCs from these mice also had impaired ability to convert naïve T cells into Th17 cells
− Importantly, mice lacking avb8 on DCs showed near-complete protection from experimental autoimmune encephalomyletis.
• Study showed that avb8 mediated TGF-B activation by DCs can only induce anti-specific T cells to become Th17 cells if the same ell that is expressing the integrin is also actively presenting antigen in the context of MHC-II.
• Since TGFB signaling can direct the formation of proinflammatory Th17 cells or TRegs, makes sense for this process to be regulated in space and time. Their results suggest that precise spatial regulation of delivery of active TGF-B allows the same DCs to provide active TGF-B and polarizing cytokines (eg, IL-6 for Th17) → giving precise control of T cell fate.
- In addition to immune function in the intestine, avb8-mediated TGF-B activation inducing Th17 cells also has a role in inflammatory disorders of the airway.
- Associated with airway hyper-respoinsiveness in allergic asthma → IL-17 induces the NF-kB/RhoA/ROCK2 pathway which increases smooth muscle contractility.
- Mice lacking avb8 on DCs show reduced AHR characterized by a marked reduction in Th17 cells in the lung
- Therefore has been suggested that pharmacological intervention of avb8 expression in the lung may be a therapeutic target for inflammatory airway disease.
- Highlights a need to gain better understanding of the biological outcomes controlled by avb8-mediated TGF-B activation, as not only can it aid understanding of the pathogensis of disease, it has the potential to assist with the development of novel therapeutics.
How are TRegs generated?
• T cells undergo checks during development.
• T cells that express a TCR for non-self antigen survive and leave the thymus
• T cells that express a TCR for self antigen and bind strongly undergo apoptosis (negative selection).
• T cells that express a TCR for self-antigen and bind with moderate affinity become natural TRegs.
− TRegs are characterized by the expression of Foxp3, essential for their suppressive function (Fontenot et al, 2003).
− Mutation of Foxp3 results in severe autoimmunity and multi-organ inflammation.
• However, negative selection isn’t perfect, and some T cells recognizing self-antigen do leave the thymus and can potentially cause inflammatory disease.
• TRegs can therefore be induced in the periphery from naïve CD4+ T cells by the de novo induction of Foxp3.
− Has been shown that a high proportion of TRegs are induced by specific commensal bacteria of the Clostridium family
• Since the discovery of both n and iTregs, there has been focus in identifying markers that can distinguish them – would allow for a better understanding of their specific functions
− Helios proposed marker of nTRegs (Thornton et al, 2010)
List the 4 ways that TRegs can suppress
- Cytokine dependent → TGF-B and IL-10
- Induction of effector T cell death → secrete perforin and granzyme
- Disruption of effector T cell metabolism → CD25 upregulated on activation, mops up IL-2 so isn’t used for effector T cell proliferation
- Targeting of DCs → CTLA-4 binds to CD80 and CD86 on DCs, downregulating these costimulatory molecules
What is the model used for studying how TRegs regulate intestinal immunity?
Adoptive Transfer Model of Colitis – for testing how TRegs mediate intestinal immunity (Mottett et al,):
• Purify naïve T cells and inject them into lymohocyte deficient mouse → develop spontaneous colitis
• Infuse TRegs at the same time, you prevent the naïve CD4_ T cells from causing the inflammation
Outline the experiments that showed that TGFB plays a role int the suppressive function of TRegs.
• In adoptive transfer model, if you take naïve T cells + TRegs → no inflammation
• Take naïve T cells that lack the TGFB receptor → TRegs can no longer suppress the inflammation
➢ Therefore, T cells need to see TGFB in order to be suppressed
• TRegs produce a lot of TGFB, however if you take TRegs that cant make TGFB and inject these – they can still suppress
• If you take these TRegs along with an antibody blocking TGFB function – they can no longer suppress
➢ Therefore, although TGFB is crucial for TReg suppression of T cells, TRegs are not the important source
➢ Hypothesised that TRegs are specialized to activate TGFB, rather than produce it
