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Intestinal permeability and autoimmune diseases

Intestinal permeability and autoimmune diseases BioscienceHorizons Volume 10 2017 10.1093/biohorizons/hzx015 ............................................................................................ ..................................................................... Review article Megan Ciara Smyth University of Glasgow, D43 David Bomberg House, 282-302 Borough High Street, SE1 1JJ, UK *Corresponding author: University of Glasgow, D43 David Bomberg House, 282-302 Borough High Street, SE1 1JJ, UK. Email: megan.smyth@hotmail.com. Supervisor: Michael Lucas, R311 Level 3, West Medical Building, University of Glasgow, G12 8QQ, UK. Michael.Lucas@glasgow.ac.uk ............................................................................................ ..................................................................... Exact aetiology of most autoimmune diseases is unknown. Genetic predisposition, environmental factors, microbiota dysbio- sis and the gut–brain axis are known to interplay in autoimmune disease development. Arresting such interplay, by imple- menting a particular diet (such as the low FODMAP diet) or by consuming specific drugs (such as zonulin antagonists) for example, will reduce disease symptoms, reverse intestinal hyperpermeability and allow remission. The aim of this study was to investigate possible mechanisms of autoimmune disease aetiology and alterations in intestinal permeability, specifically in coeliac disease and type 1 diabetes mellitus. This was done by collecting researched evidence from journals and other publications. Understanding the pathology of the diseases and identifying the particular genes and triggers involved as well as improving investigative methods will potentiate the development of prevention and treatment therapies. Methods: Collection of researched evidence was conducted from journals and other publications. Key words: intestine, permeability, autoimmune, coeliac, diabetes, zonulin Submitted on 31 January 2017; editorial decision on 7 August 2017 ............................................................................................ ..................................................................... to certain autoimmune diseases, suggesting that common Introduction immunological pathways may be involved. The observation that CTLA4 and PTPN22 are associated with various auto- When the body’s ‘great protector’, the immune system, goes immune diseases is consistent with the hypothesis that certain amiss, autoimmunity, the state in which the body attacks immunological pathways are common in multiple diseases itself, ensues. Autoimmune diseases are characterized by tis- whereas others are specific to a particular disease (Rioux and sue damage and loss of function arising from abnormal Abbas, 2005). Identifying a particular gene associated with a immune response directed against specific organs. Such dis- disease allows immunologists to investigate and better under- eases are chronic and debilitating, brutally reducing quality of stand the pathways and molecular mechanisms, vital for devel- life. More than 80 types of autoimmune disorders are known oping a cure. However, genetic predisposition only accounts to exist and a rise in both incidence and prevalence of the disor- for 10–40% of cases suggesting environmental factors and ders has induced increased scientific interest. Although aetiology microbiota health play greater roles. of autoimmune diseases is unknown, genetic predisposition, environmental factors, gut microbiota dysbiosis and defective Chemical toxins, bacteria, viruses, emotional stress and bidirectional gut–brain communication have been identified as drugs are environmental triggers potentially leading to auto- possible contributors (Campbell, 2014). immune disorders. Metals such as aluminium hydroxide, Several genes encoded by the major histocompatibility nickel and cobalt may induce inflammation by activation of complex (MHC) have been identified to predispose individuals pattern recognition receptors. Aluminium hydroxide used in ............................................................................................... .................................................................. © The Author 2017. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Review article Bioscience Horizons � Volume 10 2017 ............................................................................................... .................................................................. vaccines (for example, the hepatitis B vaccine) and medical Tight junctions silicones used in breast implants have shown to elicit The intestinal epithelium is the largest mucosal surface pre- Shoenfeld’s syndrome (Hamza et al., 2012, Vera-Lastra et al., sent in the human body (~7.5 m) and provides an interface 2012). However, environmental factors are not necessarily between the internal and external environments. Comprising the inducers but may cause amplification of an existing auto- of a single layer of epithelial cells interconnected by dynamic immune disease or cause relapse (Campbell, 2014). intercellular junctions along their lateral margins, the epithe- The inducer of autoimmune responses related to the gut is lium orchestrates the locomotor, digestive, absorptive, neu- most likely to be microbiota dysbiosis leading to increased roendocrinological and immunological functions of the intestinal permeability. The condition of the gut flora is influ- intestine. Exchange between the intestinal lumen and sub- enced by diet, mucosal health and the gut–brain axis. The mucosa of proteins can occur via two mechanisms: transcellu- human diet has changed substantially over the years, from lar transport (via endo-/exocytosis) and paracellular transport eating food shortly after harvest and in season with only occa- (via TJs) subject to molecular weight. Molecules sized sional consumption of meat, to eating what we want when we 10–15 Å are transported paracellularly, meaning ‘beside’ want, and in no modest amount. The food itself has been dra- cells, through modulation of TJs. Macromolecules greater matically transformed. New strains of grains engineered to than 15 Å however are too large and are not trafficked across resist pathogens and herbicides is one of many examples. the epithelial layer in the presence of competent TJs (Fasano, Everyday consumption of genetically modified or processed 2008). Malfunctioning TJs due to incorrect assembly will food is inevitable. Maintaining a wholesome and balanced therefore cause intestinal hyperpermeability with noxious diet is essential in ensuring healthy gut microbiota for correct effect. gut–brain communication and overall health. TJs are composed of integral proteins, including occludin, Over one hundred trillion microorganisms, including one claudins and junctional adhesion molecules (JAMs), and cyto- thousand different species of microbes encompassing more plasmic plaque junctional complex proteins (ZO-1, ZO-2, than 3 million genes, reside in human gut microbiota ZO-3, 7H6, symplekin and cingulin). Tight junctions are (Jandhyala et al., 2015). While an individual’s microbiota is bound to and interact with the apical actomyosin rings made unique, evolving from birth to old age as well as being envir- up of cellular cytoskeleton structures. Assembly occurs via a onmentally influenced, it realizes the same physiological func- cascade of biochemical events ultimately leading to a complex tion in all humans, that is, protection. The microbiota offers of exquisite anatomy and function. Opening and closing of protection by repressing pathogenic growth and prevention epithelial TJs is dependent on various stimuli including diet- against allergy and disease by regulating the immune system ary intake, neural and endocrine signals, and immunological (Guarner and Malagelada, 2003). It ensures the passage of factors such as inflammatory mediators and mast cells non-harmful molecules via the intestinal epithelium by pro- (Arrieta, Bistritz and Meddings, 2006). viding barrier fortification, appropriate mucous composition Many bacteria enhance permeability of the epithelial layer and apical tightening at tight junctions (TJs). by altering TJ state presumably for the benefit of their own Complex and bidirectional, the communication pathways growth. Vibrio cholerae was studied by Fasano (1999),a between the gut microbiota and the brain play an important gastroenterologist from the University of Maryland, who role in human health and disease. Mutualistically associated, uncovered the bacterium’s mechanism of increasing para- the gut hosts an environment potentiating microbiota growth, cellular permeability by the release of toxins, one of which is while microbiota maintains homoeostasis within the body named Zonula occludens toxin (ZOT), which binds to apical and overall health. Gut–brain and brain–gut communication membrane receptors on the enterocyte activating a cascade of occurs by means of neural (autonomic and enteric nervous intracellular events resulting in actomyosin contraction and systems), endocrine, metabolic and immune systems (Zhou TJ disassembly. Since it is unlikely that this pathway existed and Foster, 2015). Disruption of one such system may have for the sole benefit of bacteria, Fasano and his team specu- pathological consequences. lated that the pathway was a physiological one which bacteria had evolved to take advantage of. Several years later this was Together with the gut-associated lymphoid tissue and the proven to be correct as the human homologue, zonulin, was neuroendocrine network, the intestinal epithelial layer preserves identified using affinity-purified anti-ZOT antibodies and a the equilibrium between tolerance and immunity to non-self- chamber assay in foetal and adult human intestine (Wang antigens, preventing pathology and minimizing inflammation. et al., 2000). The physiological role of zonulin and the zonu- Permeability of this layer is therefore responsible in maintaining lin system is still in question. However, it is presumed to play this balance. Increased permeability is thought to be triggered a pivotal role in tissue morphogenesis, movement of fluid, by several parties and mechanisms such as bacterial endotoxins macromolecules and leucocytes between the intestinal lumen (acquired from bacterial infection or genetically modified and the interstitium, protection against microorganism colon- organisms) causing disruption of actomyosin networking lead- ization and pathological processes including inflammation, ing to epithelial cell damage and stress-induced zonulin eliciting autoimmunity and cancer. tight junction damage (Campbell, 2012). ............................................................................................... .................................................................. 2 Bioscience Horizons � Volume 10 2017 Review article ............................................................................................... .................................................................. Fasano and his team proposed a zonulin signalling path- currently being effectuated to contribute to the growing evi- way: zonulin activates epidermal growth factor receptor dence for the drug’s safety and efficacy. (EGFR) directly through EGFR-binding or indirectly via transactivation of EGFR (Zonulin binds to protease-activated receptor 2 (PAR2) followed by Src or matrix metalloprotei- Results nase/a disintegrin and metalloproteinase (MMPs/ADAMs) Coeliac disease mediated Pro-HB-EGF activation which in turn activates EGFR). EGFR activates phosphokinase C (PKC) which Coeliac disease is an autoimmune enteropathy resulting from hydrolyses phosphatidylinositol (PPI) releasing inositol 1,4,5- inappropriate T-cell-mediated immune responses to ingested trisphosphate (PPI-3) and diacylglycerol (DAG). DAG acti- gluten contained in grains such as wheat, barley and rye. vates PKCα directly by binding to it while PPI-3 indirectly Gliadin is the toxic component of gluten and is responsible 2+ activates PKCα via intracellular Ca release. Activated PKAα for intestinal damage. In the continuous presence of gliadin, catalyses the phosphorylation of target proteins ensuing poly- the disease is self-perpetuating. Other coeliac disease symp- merization of G-actin and F-actin. This causes the actin fila- toms include autoimmune targeting of tissue transglutami- ments to rearrange which displaces junctional complex nase (tTG) and association with the major histocompatibility proteins such as ZO-1 and ZO-2 loosening the TJ and allow- complex (MHC) haplotypes. Coeliac disease is strongly asso- ing the exchange of appropriately sized molecules. Once ciated with the human leucocyte antigen (HLA) gene with zonulin signalling is ceased, the TJ returns to its closed base- over 90% of patients carrying the HLA-DQ2 haplotype while line state. the remainder carry HLA-DQ8 (Lammers et al., 2008). Zonulin is the only described physiological modulator of Under normal physiological conditions, access of gliadin intercellular TJ arrangement in humans. Deregulation or dis- to submucosal tissue is prevented by competent TJs limiting ruption of the zonulin signalling pathway in genetically sus- passage of macromolecules. In individuals carrying the HLA- ceptible individuals is hypothesized to lead to increased DQ2 or -DQ8 haplotypes however, TJs are disassembled intestinal permeability and possible autoimmune disorders. allowing permeation of gliadin and triggering gliadin-induced The classic paradigm of autoimmune disorders, involving spe- immune responses eventually leading to pathology associated cific gene makeup, microbiota dysbiosis, incorrect communi- with coeliac disease (Lammers et al., 2008). Autoimmunity is cation between innate and adaptive immunity and exposure believed to be triggered via a succession of events following to environmental factors, has had to make room for a new gluten ingestion. Upon reaching the intestinal lumen, gliadin element, that of loss of junction of the intestinal barrier due to and its immunomodulatory and inflammatory fragments bind TJ displacement. Intestinal hyperpermeability is often found to chemokine C-X-C receptor 3 (CXCR3) inducing myeloid to precede autoimmune disorders and is suspected to be the differentiation primary-response protein 88 (MyD88) means by which abnormal passage of antigens from the intes- dependent zonulin release resulting in disassembly of TJs. tinal lumen results in an autoimmune response. This novel Following removal of the functional amide group by tTG, theory is encouraging as it implies autoimmunity is non- gliadin peptides, having crossed the epithelium through autoperpetuating and suggests the possibility of prevention or opened TJs, bind to HLA molecules on the antigen-presenting treatment of autoimmune disorders by arresting the interplay cell surface (APC). This triggers the release of My88- between genes and environmental triggers through intestinal dependent Zonulin and cytokines. HLA-gliadin peptide com- barrier function re-establishment, a new and innovative plexes presented by APCs cause T-lymphocyte activation of approach to treatment (Fasano, 2012a). both humoral (B cell activation leading to plasma cell release of anti-gliadin antibodies (AGA), N-arachidonoylethanola- Safety and efficacy testing of zonulin inhibitor, larazotide mide (AEA) and anti-tissue transglutaminase (αtTG) and cell- acetate (AT-1001), are currently being carried out. Results mediated (natural killer cells which destroy epithelial cells via from experiments using BioBreeding diabetes-prone (BBDP) cytokine release) responses. rats prove promising. Larazotide acetate administration to BBDP rats prevents disrupted intestinal barrier function, The interplay between the two responses (innate and adap- autoantibody production, pancreatic islet destruction and dis- tive) ultimately leads to the autoimmune process leading to ease development (70% decrease in diabetes incidence) coeliac disease. Once gluten is no longer part of the indivi- (Watts et al., 2005, Fasano, 2011). It has also shown to be duals diet, gliadin is no longer present, zonulin blood levels affective in managing coeliac disease. In an experiment car- decrease and TJs become competent once again. The intes- ried out by Leffler et al. (2015), coeliac disease suffers orally tinal epithelium resumes its normal functioning, autoantibody administered with larazotide acetate experienced reduced levels return to normal, the autoimmune response is shut-off symptoms within weeks of administration (50% reduction in and consequently, intestinal repair occurs and thus complete abdominal pain reported). This inhibitor is the first of a novel remission ensues. class of agents targeting permeability through TJ regulation In an experiment by Fasano and his team (Fasano, 2012b) and may soon be considered an important therapeutic option intended to measure possible zonulin perturbation during for autoimmune disease sufferers. More experiments are ............................................................................................... .................................................................. 3 Review article Bioscience Horizons � Volume 10 2017 ............................................................................................... .................................................................. coeliac disease, intestinal tissue was obtained from seven indi- Hashimoto’s thyroiditis, autoimmune hepatitis and connect- viduals suffering from coeliac disease and six non-sufferers. ive tissue diseases (Ventura, Magazzù and Greco, 1999). In a These were probed for zonulin with anti-zonula occludens model investigating BioBreeding rats, spontaneously develop- toxin (ZOT) antibodies. Immunofluorence studies and quan- ing autoimmune diabetes, lymphocytic destruction of islet titative immunoblotting of coeliac diseased tissues showed β-cells and other autoimmune features developed after 3–4 increased zonulin expression and higher zonulin protein con- weeks after the induction of increased intestinal permeability. centrations in the intestinal submucosa which was absent in Notably, increased permeability appeared before the develop- control tissues. Since zonulin levels have been found to be ele- ment of diabetes. Rats fed on a hydrolysed casein diet were vated in individuals with coeliac disease in the acute phase, less prevalent to diabetes development, however, their intes- when TJs are open, this suggests zonulin as the key mediator tinal permeability was not altered suggesting that it is an event in the pathology of coeliac disease. Further, an increase in preceding the disease (Meddings et al., 1999). Similarly, in a expression as a result of TJ displacement may provide study carried out on children suffering from coeliac disease, increased zonulin presentation to immune cells in the sub- increased intestinal permeability and abnormal TJ structure mucosa thus resulting in an autoimmune response. To verify was present and not entirely reversible following gluten- this hypothesis, ZOT-based ELISA was used to measure free diet implementation. These results suggest either irrevers- zonulin antibody levels in the blood of coeliac disease suf- ible damage is made to the intestinal epithelium during coeliac ferers against a control group. While anti-zonulin IgG levels disease, or alteration in TJ complexes before disease onset were similar in patients with coeliac disease and in controls, thus eliminating the disease will not eliminate the prior symp- anti-zonulin IgA levels were elevated in 21% of coeliac dis- toms (Schulzke et al., 1998). To determine the correct explan- eased patients during the acute phase of the disease. In remis- ation however, one would need to examine individuals prior sion, individuals’ anti-zonulin IgA levels and intestinal to the onset of the disease, however identifying such indivi- permeability returned to normal after 3–6 months on a duals proves challenging. gluten-free diet, further evidencing zonulin as the main con- Epithelial dysfunction prior to and during coeliac disease is tributor to disrupted barrier function. restricted to the jejunum and distal ileum, while the colon Irish setter dogs were used in an experiment to provide evi- permeability remains unperturbed (where the presence of dence for increased intestinal permeability participating in the microflora and byproducts could be detrimental upon com- aetiology of coeliac disease (Hall and Batt, 1991). The litters promised epithelium). This coincides with the regional distri- were bred from gluten-sensitive enteropathy suffering dogs. bution of ZOT receptors, with the ZOT-induced F-actin The experiment compared litters predisposed to disease redistribution in the villi, and with the detection of immuno- reared on a gluten-free diet and on a normal diet, compared fluorescent particles within the gastrointestinal tract studied to a control group of healthy dogs on a normal diet. in rabbit intestine (Fasano et al., 1997). Similar results have Permeability was found to have risen in the litters on a gluten- been described in human intestine (Uzzau et al., 2001). free diet with unaffected intestinal integrity. In litters reared Decreasing ZOT receptors along the villus-crypt axis sug- on a normal diet, increased intestinal permeability was noted gests upregulation during enterocyte differentiation of ZOT compared to that in gluten-free diet litters after just 4 months receptors. and elicited villus degeneration, intraepithelial lymphocyte By chance, Fasano (2011) uncovered the key to unlocking infiltration and reduced ALP activity. The findings support the means of oral delivery of large-molecule drugs across the the hypothesis that intestinal hyperpermeability is a primary intestinal epithelial layer. Earlier attempts to open up TJs defect involved in eliciting gluten-sensitive enteropathy in between epithelial cells failed due to the complete and irre- Irish setter dogs. It is important to note however, that the versible destruction of the TJ, differing from zonulin’s valu- gluten-free diet may have contained other elements potentiat- able reversible displacing effect. In early studies, Fasano ing sensitivity. Carrageenan is an additive often found in dog showed ZOT’s ability to induce a 10-fold increase in insulin food and is found to cause gastrointestinal inflammation, absorption and six-fold increase in absorption of immunoglo- increased susceptibility to intestinal lesions, ulcerations and bulins in rabbit small intestine (Fasano and Uzzau, 1997). tumours (Tobacman, 2001). For more pertinent results, the Diabetic rats were also studied, where ZOT administration litters could be reared on an elemental diet, a liquid diet in demonstrated a greater decreased in blood glucose levels com- which only the basic nutrients the body requires are ingested, pared to those derived following insulin injections. This opens in order to eliminate other hyperpermeability potentiating ele- up the possibility of replacing painful insulin injections with ments. Thus increased intestinal permeability is most likely oral pill ingestion. The oral delivery of drugs such as taxol not the primary defect, but rather, secondary to the presence and growth hormone in conjunction with zonulin could, for of a dietary elements potentiating increased intestinal perme- example, be considered for treating cancer. The discovery ability (Hall and Batt, 1991). Regardless, the experiment may also change the lives of many autoimmune disease suf- strongly suggests hyperpermeability precedes disease. ferers. Knowing that increased permeability of TJs predis- The opening of TJ in coeliac disease if left untreated has poses individuals to autoimmune diseases suggests that with been reported to increase chances of other autoimmune dis- appropriate permeability reinstated, likelihood of disease ease development such as type I diabetes mellitus, development is reduced. ............................................................................................... .................................................................. 4 Bioscience Horizons � Volume 10 2017 Review article ............................................................................................... .................................................................. The discovery of zonulin, a potential tool for drug and pep- A study in humans revealed early introduction of gluten- tide transport through the intestinal epithelium, is tremen- containing grains to infants increased risk of diabetes (Norris dously exciting. ZOT displays many useful properties making et al., 2003). This study looked at 900 infants with increased it a suitable candidate for the job: it is non-cytotoxic; it does T1D risk, defined by exhibiting the T1D-susceptible HLA not irreversibly destructs the epithelium, but rather, displaces gene or by having a first-degree relative with T1D. The infants TJs in a reversible manner; it interacts with a region-specific were tested at 9, 15 and 24 months and annually thereafter receptor (specific to jejunum and distal ileum); it is not effect- for insulin autoantibodies, glutamate decarboxylase (GAD) ive in the colon where it could potentiate harm via intestinal autoantibodies and islet antigen-2 (IA-2) autoantibodies by barrier disruption; it does not induce acute systemic side means of a blood test. Testing positive in at least one of these effects; and it acts relatively quickly (increased permeability autoantibodies or having diabetes itself meant testing positive detected 20 min after oral administration). for islet autoimmunity. The results showed that infants exposed to gluten between ages 0–3 months and 7+ months resulted in an elevated risk of T1D development while expos- Type I diabetes mellitus ure to the cereal between 4 and 6 months resulted in a reduced or delayed risk of T1D development. Curiously, this may indi- Type 1 diabetes mellitus (T1D) is defined by hyperglycaemia cate a window of exposure to gluten in infancy outside of instigated by an autoimmune destruction of insulin- which islet autoimmunity and T1D risk in susceptible infants producing β-cells in the pancreas. Various genes have been is increased. The study also found that if gluten-containing identified to be involved in T1D susceptibility. The main two grains were introduced while the infant was still breastfeed- are the HLA gene region IDDM1, located on chromosome ing, the risk of islet autoimmunity and T1D was reduced, 6p21 and accounting for 42% of genetic susceptibility, and independent of the window of exposure previously the insulin gene region IDDM2, on chromosome 11p account- calculated. ing 10% of genetic susceptibility. Other known chromosomal Administration of antibiotics such as doxycycline, fusidic locations with undetermined susceptibility genes have been acid, colistin and Bactrim to BBDP rats and NOD mice after described (Kelly et al., 2003). Additionally, polymorphisms in weaning leads to the prevention of T1D development. MYO9B, a gene involved in intestinal permeability have also Although the mechanism is not entirely understood, micro- shown interrelation with T1D development (Santiago et al., biota dysbiosis affects the development of T1D in both animal 2008). models. This is further supported by a study administrating Subjected to particular environmental triggers, genetically probiotics to NOD mice which was found to induce IL-10 predisposed individuals may develop T1D. Such triggers have production, an immunoregulator and anti-inflammatory cyto- yet to be identified, and would allow for prevention and treat- kine, and to prevent T1D development (Calcinaro et al., ment of the disease. Gliadin is believed to be a T1D trigger, its 2005). Another study revealed neonatal administration of an mechanism similar to that in coeliac disease. Non obese dia- immunodominant epitope of T1D, peptide p277, proved to betic (NOD) mice and BBDP rats have been used to demon- potentiate the protective effect of a hydrolysed casein diet, strate that gliadin is a dietary trigger for T1D showing reducing the incidence of T1D by 64% or delaying its devel- increased intestinal permeability following wheat administra- opment (Brugman et al., 2004). tion. Rearing NOD mice and BBDP rats on a gluten-free diet The jejunum of T1D sufferers have been found to display resulted in delayed and reduced T1D development. The study increased IFNγ and TNF-α,inflammatory response factors also showed that time of exposure to the trigger is important: leading to pancreatic damage, their amounts correlating with delayed gluten exposure by prolonging breastfeeding led to the severity of the disease (Westerholm-Ormio et al., 2003). reduced T1D development in BBDP rats (Visser et al., 2003). Significantly, a Th1 bias has been found in the mediastinal The experiment investigated the relationship between dur- lymph node (MLN), a gut-associated secondary lymphoid ation of exclusive breastfeeding and the onset of T1D. organ where APCs present soluble dietary antigens to naïve T Normal rat breastfeeding duration is 21 days, in this experi- cells, of T1D individuals (Chakir et al., 2005). In healthy indi- ment however the lactation period was prolonged to 31 and viduals, the gut mucosa is a Th2 predominant environment 40 days. In BBDP rats on a 31 day breastfeeding diet, reduced which allows for normal immunosuppressive response to glu- onset of T1D was noted compared to that of the 21 day ten. In T1D rats, however, a Th1 bias is apparent, resulting breastfeeding control group. Surprisingly, rats on a 40 day from a Th2-specific Gata3 deficit causing an imbalance in breastfeeding diet showed unchanged risk of T1D and were just Th1/Th2 differentiation. Elevated Th1 levels results in as prone to T1D as the control rats. These rats were underweight increased IFNγ and TNF-α production leading to pancreatic andexhibited reducedgrowthratecharacteristic of malnutrition, islet β-cell damage. Chakir and his team also found gluten- known to reduce β-cell function in later-life. Poor functioning of specific CD4+ T cells in the MLN of early-stage diabetic rats. β-cells impairs the insulin-secreting capacity of the pancreas. The Low frequencies of the cells were found in the spleen, suggest- study draws the conclusion that while increased exclusive breast- ing activation of CD4+ T cell in the gut. This experiment links feeding period may reduce T1D risk, excessive prolongation of the gut immune system and T1D. this period may annul the positive effect. ............................................................................................... .................................................................. 5 Review article Bioscience Horizons � Volume 10 2017 ............................................................................................... .................................................................. Bosi et al. (2006) showed increased intestinal permeability gene expression in intestinal mucosal cells following exposure to precede T1D. He studied 81 subjects with islet autoimmun- to gliadin in T1D individuals demonstrating intestinal T-cell ity at varying stages (preclinical, newly-onset and long-term activation by gliadin (Auricchio et al., 2004). established diabetes) and 40 control subjects and submitted In conclusion, individuals with T1D are either genetically them to the lactulose–mannitol test. Orally administered, the predisposed, have been subjected to environmental triggers or two sugars were measured in urine to extrapolate intestinal experienced gut microbiota dysbiosis and display increased permeability. Disaccharide lactulose, a larger molecule, intestinal permeability, heightened immune activation (e.g. crosses the intestinal epithelium intracellularly via TJs while inflammation), histological changes and elevated plasma monosaccharide mannitol, a smaller molecule, is transported zonulin. In contrast to coeliac disease with the sole trigger of across the epithelium transcellularly through water-filled gliadin, T1D is believed to have several environmental trig- pores on the cell membrane. Altered intestinal anatomy or gers. Bovine insulin, in cow milk, has been shown to elicit functional integrity would result in facilitated passage of one similar reactions to gliadin resulting in T1D development. or both molecules. Intestinal permeability to lactulose was Bovine insulin administration in infants may sensitize intes- shown to be elevated in all islet autoimmunity individuals tinal T-cells later partaking in pancreatic insulin-producing β- indicating barrier damage while integral surface mucosa cell destruction, an autoimmune response. Evidently further remaining intact shown by unchanged permeability to manni- research is necessary wholly to understand the role of the gut tol compared to controls, thus increasing the lactulose:mani- and its microbiota in T1D. However, it is understood that tol ratio. This study supports the hypothesis that increased microbiota dysbiosis causes upregulation of gut and systemic intestinal permeability precedes T1D. inflammation, due to endotoxins from pathogenic bacteria or The morphological aspect of the intestinal mucosa in non- to pro-inflammatory cytokines synthesized by immune cells, coeliac T1D individuals was studied by Secondulfo et al. and causes T1D as well as disrupting gut–brain axis commu- (2004) to investigate reasons for increased permeability. Four nication potentially leading to neuroinflammation and cogni- parameters of the mucosal ultra-structural were evaluated: tive dysfunction (e.g. Alzheimer’s) (Bhattacharjee and Lukiw, microvilli height, microvilli thickness, distance between 2013). Identifying the particular environmental triggers and microvilli, and TJ thickness. Reduced density, displacement improving investigative methods will greatly help evaluate the and abnormal (short and thin) formation of microvilli were intestinal epithelium and hopefully provide important insights found on the apical membrane enterocytes, many of which towards better understanding the pathogenic process of the contained large vacuoles, engorged mitochondria and a sur- disease and consequentially potentiating prevention and plus of lysosomes. Enterocyte intercellular spaces were treatment. enlarged and TJ domains were displaced and thickened. The disturbed intestinal epithelium function in non-coeliac T1D Discussion individuals was associated with altered mucosal morphology suggesting loss of function of the intestinal barrier leading to As evidenced, autoimmune disorders develop over time and pathogenesis. can be preclinically detected. Due to their ambiguous symp- A wide collection of studies demonstrate synergism toms (fatigue, muscle or joint ache, general malaise), correct between microbiota dysbiosis, increased intestinal permeabil- diagnosis of autoimmune disorders can prove difficult. Many ity, altered mucosal immunity and pathogenesis of T1D. autoimmune disease sufferers wait until symptoms aggravate Kuitunen et al. (2002) found an increased lactulose and man- and the disease begins to take its toll before seeking medical nitol absorption in T1D sufferers thought to be due to altered advice, at which stage disease reversal may no longer be intestinal permeability (Kuitunen et al., 2002, Secondulfo possible. et al., 2004, Sapone et al., 2006). Increase in absorption of The majority of autoimmune disorders are not well under- the two sugars was also found to be the case for β-cell auto- stood. Identification of the triad of triggers—genes, environ- immune individuals suggesting altered intestinal permeability mental factors and microbiotia dysbiosis—as well as prior to the onset of T1D (Bosi et al., 2006). Histological improvements of investigative techniques will allow better changes in height and thickness of intestinal villi as well as understanding of the pathological processes involved and per- their spacing and TJ assembly was altered in T1D individuals mit treatment development. Understanding such functioning signifying the presence of mucosal injury (Secondulfo et al., will allow prevention of disordered intercellular communica- 2004). Structural changes in TJ complexes in T1D were tion and digestive disorders leading to autoimmune diseases, understood from recordings of elevated plasma zonulin levels tissue inflammation, harmful adaptations and metastasis. in individuals with T1D (Sapone et al., 2006). Several experi- Remarkably, the same system which can cause such damage ments have investigated intestinal biopsies in children with could also be the key to curing hundreds of diseases through T1D and found elevated interleukin and interferon levels tissue specific drug delivery. indicating intestinal inflammation (Savilahti et al., 1999, Westerholm-Ormio et al., 2003). Auricchio and his team Fasano unknowingly uncovered an innovative strategy for + + found heightened CD3 and CD25 cells and increased HLA the delivery of drugs, macromolecules and vaccines across the ............................................................................................... .................................................................. 6 Bioscience Horizons � Volume 10 2017 Review article ............................................................................................... .................................................................. body’s various epithelia in an innocuous and minimally Acknowledgements inflammatory manner. Interestingly, not only intestinal but extra-intestinal epithelia such as tracheobronchial and renal I wish to express my sincere gratitude to Dr Michael Lucas, my tubule, as well as the vascular endothelium including the supervisor, for his guidance, enthusiasm and invaluable advice blood brain barrier may be targeted. on this subject. I would also like to thank my friends and family, for their encouragement to complete this dissertation. Zonulin analogues, such as AT-1002, have been developed and promote tissue penetration of macromolecules across a wide variety of surfaces while preserving membrane integrity thus altering permeability in a non-destructive, transient and References reversible manner. Multiple delivery routes mean facilitated administration of the vaccine or drug increasing patient com- Arrieta, M. C., Bistritz, L. and Meddings, J. B. (2006) Alterations in intes- pliance and appeal to practitioners. AT-1002 has been tinal permeability, Gut, 55 (10), 1512–1520. demonstrated on nasal epithelia, intestinal epithelia and cul- Auricchio, R., Paparo, F., Maglio, M. et al. (2004) In vitro-deranged intes- tured brain endothelial cells. It can be used to transport PEG tinal immune response to gliadin in type 1 diabetes, Diabetes,53 or insulin across the intestinal epithelium for treatment of (7), 1680–1683. constipated or diabetic individuals (Fasano and Uzzau, 1997, Song, Fasano and Eddington, 2008). AT-1002 can also Bhattacharjee, S. and Lukiw, W. J. (2013) Alzheimer’s disease and the induce rapid and reversible permeability of the blood brain microbiome’, Frontiers in Cellular Neuroscience, 7, 153. barrier and thus increase absorption of doxorubicin and Bosi, E., Molteni, L., Radaelli, M. G. et al. (2006) Increased intestinal per- paclitaxel for cancer treatment (Karyekar et al., 2003). The meability precedes clinical onset of type 1 diabetes, Diabetologia, analogue also instigates protective immune responses by adju- 49 (12), 2824–2827. vant mucosal antigen delivery when administered nasally or rectally (e.g. ovalbumin and tetanus toxoid (Marinaro et al., Brugman, S., Klatter, F. A., Visser, J. et al. (2004) Neonatal oral adminis- 2003)). Cyclosporine transported with AT-1002 is used in the tration of DiaPep277, combined with hydrolysed casein diet, pro- prevention of transplant rejection, Crohn’s disease and tects against Type 1 diabetes in BB-DP rats. An experimental study, rheumatoid arthritis. It has also been experimentally tested to Diabetologia, 47 (7), 1331–1333. treat aids (AT-1002 taken along with ritonavir and saquina- vir) and viruses such as varicella, herpes genitalis and zoster Calcinaro, F., Dionisi, S., Marinaro, M. et al. (2005) Oral probiotic admin- (with acyclovir) (Salama et al., 2005). istration induces interleukin-10 production and prevents spontan- eous autoimmune diabetes in the non-obese diabetic mouse, To summarize, zonulin agonists can be used to reversibly Diabetologia, 48 (8), 1565–1575. increase paracellular transport of drug delivery with less tox- icity than previous absorptive enhancers, while antagonists Campbell, A. W. (2012) Organic vs conventional, Alternative Therapies in can be used to prevent increased absorption of pathogenic Health and Medicine, 18 (6), 8–9. molecules or allergens. Zonulin agonists and antagonists have Campbell, A. W. (2014) Autoimmunity and the gut, Autoimmune the potential for significant and effective use in pharmacology Disease, 2014, 152428. not only in autoimmune disorders but in all diseases. Chakir, H., Lefebvre, D. E., Wang, H. et al. (2005) Wheat protein-induced In conclusion, we know significantly more today than we proinflammatory T helper 1 bias in mesenteric lymph nodes of did 5 years ago and it is to be hoped that we may be able to young diabetes-prone rats, Diabetologia, 48 (8), 1576–1584. say the same in 5 years’ time. The non-destructive opening of tight junctions means ‘open sesame’ to new therapies. Large Fasano, A. (1999) Cellular microbiology: can we learn cell physiology long-term studies, however, are required to determine poten- from microorganisms? The American Journal of Physiology, 276 (4 Pt tial adverse effects of possible new therapeutic approaches. So 1), C765–C776. far, the results look promising. Since it is now known that Fasano, A. (2008) Physiological, pathological, and therapeutic implications hyperpermeability precedes disease, a new and innovative of zonulin-mediated intestinal barrier modulation: living life on the approach to treatment, arresting the interplay between genes edge of the wall, The American Journal of Pathology, 173 (5), 1243–1252. and environmental factors through intestinal barrier function re-establishment, can be used in prevention or treatment of Fasano, A. (2011) Zonulin and its regulation of intestinal barrier func- autoimmune disorders. tion: the biological door to inflammation, autoimmunity, and can- cer, Physiological Reviews, 91 (1), 151–175. Author biography Fasano, A. (2012a) Leaky gut and autoimmune diseases, Clinical Reviews in Allergy & Immunology, 42 (1), 71–78. Graduated in 2016 with a First Class Honours in Physiology Fasano, A. (2012b) Zonulin, regulation of tight junctions, and auto- (BSc) from the University of Glasgow. Currently studying immune diseases, Annals of the New York Academy of Sciences, Radiotherapy & Oncology (MSc) at London South Bank 1258, 25–33. University, graduating in 2018. ............................................................................................... .................................................................. 7 Review article Bioscience Horizons � Volume 10 2017 ............................................................................................... .................................................................. Fasano, A. and Uzzau, S. (1997) Modulation of intestinal tight junctions Rioux, J. D. and Abbas, A. K. (2005) Paths to understanding the genetic by Zonula occludens toxin permits enteral administration of insulin basis of autoimmune disease, Nature, 435 (7042), 584–589. and other macromolecules in an animal model, The Journal of Salama, N. N., Fasano, A., Thakar, M. et al. (2005) The impact of DeltaG Clinical Investigation, 99 (6), 1158–1164. on the oral bioavailability of low bioavailable therapeutic agents, Fasano, A., Uzzau, S., Fiore, C. et al. (1997) The enterotoxic effect of The Journal of Pharmacology and Experimental Therapeutics, 312 (1), zonula occludens toxin on rabbit small intestine involves the para- 199–205. cellular pathway, Gastroenterology, 112 (3), 839–846. Santiago, J. L., Martínez, A., Núñez, C. et al. (2008) Association of Guarner, F. and Malagelada, J. R. (2003) Gut flora in health and disease, MYO9B haplotype with type 1 diabetes, Human Immunology, 69 (2), Lancet, 361 (9356), 512–519. 112–115. Hall, E. J. and Batt, R. M. (1991) Abnormal permeability precedes the Sapone, A., de Magistris, L., Pietzak, M. et al. (2006) Zonulin upregula- development of a gluten sensitive enteropathy in Irish setter dogs, tion is associated with increased gut permeability in subjects Gut, 32 (7), 749–753. with type 1 diabetes and their relatives, Diabetes, 55 (5), 1443–1449. Hamza, H., Cao, J., Li, X. et al. (2012) Hepatitis B vaccine induces apop- totic death in Hepa1-6 cells, Apoptosis: An International Journal on Savilahti, E., Ormälä, T., Saukkonen, T. et al. (1999) Jejuna of patients Programmed Cell Death, 17 (5), 516–527. with insulin-dependent diabetes mellitus (IDDM) show signs of immune activation, Clinical and Experimental Immunology, 116 (1), Jandhyala, S. M., Talukdar, R., Subramanyam, C. et al. (2015) Role of the 70–77. normal gut microbiota, World Journal of Gastroenterology: WJG,21 (29), 8787–8803. Schulzke, J. D., Bentzel, C. J., Schulzke, I. et al. (1998) Epithelial tight junction structure in the jejunum of children with acute and treated Karyekar, C. S., Fasano, A., Raje, S. et al. (2003) Zonula occludens tox- celiac sprue, Pediatric Research, 43 (4 Pt 1), 435–441. in increases the permeability of molecular weight markers and chemotherapeutic agents across the bovine brain microvessel Secondulfo, M., Iafusco, D., Carratù, R. et al. (2004) Ultrastructural muco- endothelial cells, Journal of Pharmaceutical Sciences,92(2), sal alterations and increased intestinal permeability in non-celiac, 414–423. type I diabetic patients, Digestive and Liver Disease: Official Journal of the Italian Society of Gastroenterology and the Italian Association Kelly, M. A., Rayner, M. L., Mijovic, C. H. et al. (2003) Molecular aspects of for the Study of the Liver, 36 (1), 35–45. type 1 diabetes, Molecular Pathology: MP, 56 (1), 1–10. Song, K. H., Fasano, A. and Eddington, N. D. (2008) Enhanced nasal Kuitunen, M., Saukkonen, T., Ilonen, J. et al. (2002) Intestinal permeabil- absorption of hydrophilic markers after dosing with AT1002, a tight ity to mannitol and lactulose in children with type 1 diabetes with junction modulator, European Journal of Pharmaceutics and the HLA-DQB1*02 allele, Autoimmunity, 35 (5), 365–368. Biopharmaceutics: Official Journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e.V, 69 (1), 231–237. Lammers, K. M., Lu, R., Brownley, J. et al. (2008) Gliadin induces an increase in intestinal permeability and zonulin release by binding Tobacman, J. K. (2001) Review of harmful gastrointestinal effects of car- to the chemokine receptor CXCR3, Gastroenterology, 135 (1), rageenan in animal experiments, Environmental Health Perspectives, 194–204.e3. 109 (10), 983–994. Leffler, D. A., Kelly, C. P., Green, P. H. et al. (2015) Larazotide acetate Uzzau, S., Lu, R., Wang, W. et al. (2001) Purification and preliminary for persistent symptoms of celiac disease despite a gluten-free characterization of the zonula occludens toxin receptor from diet: a randomized controlled trial, Gastroenterology, 148 (7), human (CaCo ) and murine (IEC6) intestinal cell lines, FEMS 1311–1319.e6. Microbiology Letters , 194 (1), 1–5. Marinaro, M., Fasano, A. and De Magistris, M. T. (2003) Zonula occlu- Ventura, A., Magazzù, G. and Greco, L. (1999) Duration of exposure to dens toxin acts as an adjuvant through different mucosal routes gluten and risk for autoimmune disorders in patients with celiac and induces protective immune responses, Infection and Immunity, disease. SIGEP Study Group for Autoimmune Disorders in Celiac 71 (4), 1897–1902. Disease, Gastroenterology, 117 (2), 297–303. Meddings, J. B., Jarand, J., Urbanski, S. J. et al. (1999) Increased gastro- Vera-Lastra, O., Medina, G., Cruz-Dominguez, M. E. P. et al. (2012) intestinal permeability is an early lesion in the spontaneously dia- Human adjuvant disease induced by foreign substances: a new betic BB rat, The American Journal of Physiology, 276 (4 Pt 1), model of ASIA (Shoenfeld’s syndrome), Lupus, 21 (2), 128–135. G951–G957. Visser, J., Groen, H., Klatter, F. et al. (2003) The diabetes prone BB rat Norris, J. M., Barriga, K., Klingensmith, G. et al. (2003) Timing of initial model of IDDM shows duration of breastfeeding to influence Type cereal exposure in infancy and risk of islet autoimmunity, JAMA: 1 diabetes development later in life, Diabetologia, 46 (12), The Journal of the American Medical Association, 290 (13), 1711–1713. 1713–1720. ............................................................................................... .................................................................. 8 Bioscience Horizons � Volume 10 2017 Review article ............................................................................................... .................................................................. Wang, W., Uzzau, S., Goldblum, S. E. et al. (2000) Human zonulin, a Westerholm-Ormio, M., Vaarala, O., Pihkala, P. et al. (2003) potential modulator of intestinal tight junctions, Journal of Cell Immunologic activity in the small intestinal mucosa of pediatric Science, 113 (Pt 24), 4435–4440. patients with type 1 diabetes, Diabetes, 52 (9), 2287–2295. Watts, T., Berti, I., Sapone, A. et al. (2005) Role of the intestinal tight Zhou, L. and Foster, J. A. (2015) Psychobiotics and the gut-brain axis: in junction modulator zonulin in the pathogenesis of type I diabetes the pursuit of happiness, Neuropsychiatric Disease and Treatment, in BB diabetic-prone rats, Proceedings of the National Academy of 11, 715–723. Sciences of the United States of America, 102 (8), 2916–2921. ............................................................................................... .................................................................. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Bioscience Horizons Oxford University Press

Intestinal permeability and autoimmune diseases

Bioscience Horizons , Volume 10 – Nov 28, 2017

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10.1093/biohorizons/hzx015
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Abstract

BioscienceHorizons Volume 10 2017 10.1093/biohorizons/hzx015 ............................................................................................ ..................................................................... Review article Megan Ciara Smyth University of Glasgow, D43 David Bomberg House, 282-302 Borough High Street, SE1 1JJ, UK *Corresponding author: University of Glasgow, D43 David Bomberg House, 282-302 Borough High Street, SE1 1JJ, UK. Email: megan.smyth@hotmail.com. Supervisor: Michael Lucas, R311 Level 3, West Medical Building, University of Glasgow, G12 8QQ, UK. Michael.Lucas@glasgow.ac.uk ............................................................................................ ..................................................................... Exact aetiology of most autoimmune diseases is unknown. Genetic predisposition, environmental factors, microbiota dysbio- sis and the gut–brain axis are known to interplay in autoimmune disease development. Arresting such interplay, by imple- menting a particular diet (such as the low FODMAP diet) or by consuming specific drugs (such as zonulin antagonists) for example, will reduce disease symptoms, reverse intestinal hyperpermeability and allow remission. The aim of this study was to investigate possible mechanisms of autoimmune disease aetiology and alterations in intestinal permeability, specifically in coeliac disease and type 1 diabetes mellitus. This was done by collecting researched evidence from journals and other publications. Understanding the pathology of the diseases and identifying the particular genes and triggers involved as well as improving investigative methods will potentiate the development of prevention and treatment therapies. Methods: Collection of researched evidence was conducted from journals and other publications. Key words: intestine, permeability, autoimmune, coeliac, diabetes, zonulin Submitted on 31 January 2017; editorial decision on 7 August 2017 ............................................................................................ ..................................................................... to certain autoimmune diseases, suggesting that common Introduction immunological pathways may be involved. The observation that CTLA4 and PTPN22 are associated with various auto- When the body’s ‘great protector’, the immune system, goes immune diseases is consistent with the hypothesis that certain amiss, autoimmunity, the state in which the body attacks immunological pathways are common in multiple diseases itself, ensues. Autoimmune diseases are characterized by tis- whereas others are specific to a particular disease (Rioux and sue damage and loss of function arising from abnormal Abbas, 2005). Identifying a particular gene associated with a immune response directed against specific organs. Such dis- disease allows immunologists to investigate and better under- eases are chronic and debilitating, brutally reducing quality of stand the pathways and molecular mechanisms, vital for devel- life. More than 80 types of autoimmune disorders are known oping a cure. However, genetic predisposition only accounts to exist and a rise in both incidence and prevalence of the disor- for 10–40% of cases suggesting environmental factors and ders has induced increased scientific interest. Although aetiology microbiota health play greater roles. of autoimmune diseases is unknown, genetic predisposition, environmental factors, gut microbiota dysbiosis and defective Chemical toxins, bacteria, viruses, emotional stress and bidirectional gut–brain communication have been identified as drugs are environmental triggers potentially leading to auto- possible contributors (Campbell, 2014). immune disorders. Metals such as aluminium hydroxide, Several genes encoded by the major histocompatibility nickel and cobalt may induce inflammation by activation of complex (MHC) have been identified to predispose individuals pattern recognition receptors. Aluminium hydroxide used in ............................................................................................... .................................................................. © The Author 2017. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Review article Bioscience Horizons � Volume 10 2017 ............................................................................................... .................................................................. vaccines (for example, the hepatitis B vaccine) and medical Tight junctions silicones used in breast implants have shown to elicit The intestinal epithelium is the largest mucosal surface pre- Shoenfeld’s syndrome (Hamza et al., 2012, Vera-Lastra et al., sent in the human body (~7.5 m) and provides an interface 2012). However, environmental factors are not necessarily between the internal and external environments. Comprising the inducers but may cause amplification of an existing auto- of a single layer of epithelial cells interconnected by dynamic immune disease or cause relapse (Campbell, 2014). intercellular junctions along their lateral margins, the epithe- The inducer of autoimmune responses related to the gut is lium orchestrates the locomotor, digestive, absorptive, neu- most likely to be microbiota dysbiosis leading to increased roendocrinological and immunological functions of the intestinal permeability. The condition of the gut flora is influ- intestine. Exchange between the intestinal lumen and sub- enced by diet, mucosal health and the gut–brain axis. The mucosa of proteins can occur via two mechanisms: transcellu- human diet has changed substantially over the years, from lar transport (via endo-/exocytosis) and paracellular transport eating food shortly after harvest and in season with only occa- (via TJs) subject to molecular weight. Molecules sized sional consumption of meat, to eating what we want when we 10–15 Å are transported paracellularly, meaning ‘beside’ want, and in no modest amount. The food itself has been dra- cells, through modulation of TJs. Macromolecules greater matically transformed. New strains of grains engineered to than 15 Å however are too large and are not trafficked across resist pathogens and herbicides is one of many examples. the epithelial layer in the presence of competent TJs (Fasano, Everyday consumption of genetically modified or processed 2008). Malfunctioning TJs due to incorrect assembly will food is inevitable. Maintaining a wholesome and balanced therefore cause intestinal hyperpermeability with noxious diet is essential in ensuring healthy gut microbiota for correct effect. gut–brain communication and overall health. TJs are composed of integral proteins, including occludin, Over one hundred trillion microorganisms, including one claudins and junctional adhesion molecules (JAMs), and cyto- thousand different species of microbes encompassing more plasmic plaque junctional complex proteins (ZO-1, ZO-2, than 3 million genes, reside in human gut microbiota ZO-3, 7H6, symplekin and cingulin). Tight junctions are (Jandhyala et al., 2015). While an individual’s microbiota is bound to and interact with the apical actomyosin rings made unique, evolving from birth to old age as well as being envir- up of cellular cytoskeleton structures. Assembly occurs via a onmentally influenced, it realizes the same physiological func- cascade of biochemical events ultimately leading to a complex tion in all humans, that is, protection. The microbiota offers of exquisite anatomy and function. Opening and closing of protection by repressing pathogenic growth and prevention epithelial TJs is dependent on various stimuli including diet- against allergy and disease by regulating the immune system ary intake, neural and endocrine signals, and immunological (Guarner and Malagelada, 2003). It ensures the passage of factors such as inflammatory mediators and mast cells non-harmful molecules via the intestinal epithelium by pro- (Arrieta, Bistritz and Meddings, 2006). viding barrier fortification, appropriate mucous composition Many bacteria enhance permeability of the epithelial layer and apical tightening at tight junctions (TJs). by altering TJ state presumably for the benefit of their own Complex and bidirectional, the communication pathways growth. Vibrio cholerae was studied by Fasano (1999),a between the gut microbiota and the brain play an important gastroenterologist from the University of Maryland, who role in human health and disease. Mutualistically associated, uncovered the bacterium’s mechanism of increasing para- the gut hosts an environment potentiating microbiota growth, cellular permeability by the release of toxins, one of which is while microbiota maintains homoeostasis within the body named Zonula occludens toxin (ZOT), which binds to apical and overall health. Gut–brain and brain–gut communication membrane receptors on the enterocyte activating a cascade of occurs by means of neural (autonomic and enteric nervous intracellular events resulting in actomyosin contraction and systems), endocrine, metabolic and immune systems (Zhou TJ disassembly. Since it is unlikely that this pathway existed and Foster, 2015). Disruption of one such system may have for the sole benefit of bacteria, Fasano and his team specu- pathological consequences. lated that the pathway was a physiological one which bacteria had evolved to take advantage of. Several years later this was Together with the gut-associated lymphoid tissue and the proven to be correct as the human homologue, zonulin, was neuroendocrine network, the intestinal epithelial layer preserves identified using affinity-purified anti-ZOT antibodies and a the equilibrium between tolerance and immunity to non-self- chamber assay in foetal and adult human intestine (Wang antigens, preventing pathology and minimizing inflammation. et al., 2000). The physiological role of zonulin and the zonu- Permeability of this layer is therefore responsible in maintaining lin system is still in question. However, it is presumed to play this balance. Increased permeability is thought to be triggered a pivotal role in tissue morphogenesis, movement of fluid, by several parties and mechanisms such as bacterial endotoxins macromolecules and leucocytes between the intestinal lumen (acquired from bacterial infection or genetically modified and the interstitium, protection against microorganism colon- organisms) causing disruption of actomyosin networking lead- ization and pathological processes including inflammation, ing to epithelial cell damage and stress-induced zonulin eliciting autoimmunity and cancer. tight junction damage (Campbell, 2012). ............................................................................................... .................................................................. 2 Bioscience Horizons � Volume 10 2017 Review article ............................................................................................... .................................................................. Fasano and his team proposed a zonulin signalling path- currently being effectuated to contribute to the growing evi- way: zonulin activates epidermal growth factor receptor dence for the drug’s safety and efficacy. (EGFR) directly through EGFR-binding or indirectly via transactivation of EGFR (Zonulin binds to protease-activated receptor 2 (PAR2) followed by Src or matrix metalloprotei- Results nase/a disintegrin and metalloproteinase (MMPs/ADAMs) Coeliac disease mediated Pro-HB-EGF activation which in turn activates EGFR). EGFR activates phosphokinase C (PKC) which Coeliac disease is an autoimmune enteropathy resulting from hydrolyses phosphatidylinositol (PPI) releasing inositol 1,4,5- inappropriate T-cell-mediated immune responses to ingested trisphosphate (PPI-3) and diacylglycerol (DAG). DAG acti- gluten contained in grains such as wheat, barley and rye. vates PKCα directly by binding to it while PPI-3 indirectly Gliadin is the toxic component of gluten and is responsible 2+ activates PKCα via intracellular Ca release. Activated PKAα for intestinal damage. In the continuous presence of gliadin, catalyses the phosphorylation of target proteins ensuing poly- the disease is self-perpetuating. Other coeliac disease symp- merization of G-actin and F-actin. This causes the actin fila- toms include autoimmune targeting of tissue transglutami- ments to rearrange which displaces junctional complex nase (tTG) and association with the major histocompatibility proteins such as ZO-1 and ZO-2 loosening the TJ and allow- complex (MHC) haplotypes. Coeliac disease is strongly asso- ing the exchange of appropriately sized molecules. Once ciated with the human leucocyte antigen (HLA) gene with zonulin signalling is ceased, the TJ returns to its closed base- over 90% of patients carrying the HLA-DQ2 haplotype while line state. the remainder carry HLA-DQ8 (Lammers et al., 2008). Zonulin is the only described physiological modulator of Under normal physiological conditions, access of gliadin intercellular TJ arrangement in humans. Deregulation or dis- to submucosal tissue is prevented by competent TJs limiting ruption of the zonulin signalling pathway in genetically sus- passage of macromolecules. In individuals carrying the HLA- ceptible individuals is hypothesized to lead to increased DQ2 or -DQ8 haplotypes however, TJs are disassembled intestinal permeability and possible autoimmune disorders. allowing permeation of gliadin and triggering gliadin-induced The classic paradigm of autoimmune disorders, involving spe- immune responses eventually leading to pathology associated cific gene makeup, microbiota dysbiosis, incorrect communi- with coeliac disease (Lammers et al., 2008). Autoimmunity is cation between innate and adaptive immunity and exposure believed to be triggered via a succession of events following to environmental factors, has had to make room for a new gluten ingestion. Upon reaching the intestinal lumen, gliadin element, that of loss of junction of the intestinal barrier due to and its immunomodulatory and inflammatory fragments bind TJ displacement. Intestinal hyperpermeability is often found to chemokine C-X-C receptor 3 (CXCR3) inducing myeloid to precede autoimmune disorders and is suspected to be the differentiation primary-response protein 88 (MyD88) means by which abnormal passage of antigens from the intes- dependent zonulin release resulting in disassembly of TJs. tinal lumen results in an autoimmune response. This novel Following removal of the functional amide group by tTG, theory is encouraging as it implies autoimmunity is non- gliadin peptides, having crossed the epithelium through autoperpetuating and suggests the possibility of prevention or opened TJs, bind to HLA molecules on the antigen-presenting treatment of autoimmune disorders by arresting the interplay cell surface (APC). This triggers the release of My88- between genes and environmental triggers through intestinal dependent Zonulin and cytokines. HLA-gliadin peptide com- barrier function re-establishment, a new and innovative plexes presented by APCs cause T-lymphocyte activation of approach to treatment (Fasano, 2012a). both humoral (B cell activation leading to plasma cell release of anti-gliadin antibodies (AGA), N-arachidonoylethanola- Safety and efficacy testing of zonulin inhibitor, larazotide mide (AEA) and anti-tissue transglutaminase (αtTG) and cell- acetate (AT-1001), are currently being carried out. Results mediated (natural killer cells which destroy epithelial cells via from experiments using BioBreeding diabetes-prone (BBDP) cytokine release) responses. rats prove promising. Larazotide acetate administration to BBDP rats prevents disrupted intestinal barrier function, The interplay between the two responses (innate and adap- autoantibody production, pancreatic islet destruction and dis- tive) ultimately leads to the autoimmune process leading to ease development (70% decrease in diabetes incidence) coeliac disease. Once gluten is no longer part of the indivi- (Watts et al., 2005, Fasano, 2011). It has also shown to be duals diet, gliadin is no longer present, zonulin blood levels affective in managing coeliac disease. In an experiment car- decrease and TJs become competent once again. The intes- ried out by Leffler et al. (2015), coeliac disease suffers orally tinal epithelium resumes its normal functioning, autoantibody administered with larazotide acetate experienced reduced levels return to normal, the autoimmune response is shut-off symptoms within weeks of administration (50% reduction in and consequently, intestinal repair occurs and thus complete abdominal pain reported). This inhibitor is the first of a novel remission ensues. class of agents targeting permeability through TJ regulation In an experiment by Fasano and his team (Fasano, 2012b) and may soon be considered an important therapeutic option intended to measure possible zonulin perturbation during for autoimmune disease sufferers. More experiments are ............................................................................................... .................................................................. 3 Review article Bioscience Horizons � Volume 10 2017 ............................................................................................... .................................................................. coeliac disease, intestinal tissue was obtained from seven indi- Hashimoto’s thyroiditis, autoimmune hepatitis and connect- viduals suffering from coeliac disease and six non-sufferers. ive tissue diseases (Ventura, Magazzù and Greco, 1999). In a These were probed for zonulin with anti-zonula occludens model investigating BioBreeding rats, spontaneously develop- toxin (ZOT) antibodies. Immunofluorence studies and quan- ing autoimmune diabetes, lymphocytic destruction of islet titative immunoblotting of coeliac diseased tissues showed β-cells and other autoimmune features developed after 3–4 increased zonulin expression and higher zonulin protein con- weeks after the induction of increased intestinal permeability. centrations in the intestinal submucosa which was absent in Notably, increased permeability appeared before the develop- control tissues. Since zonulin levels have been found to be ele- ment of diabetes. Rats fed on a hydrolysed casein diet were vated in individuals with coeliac disease in the acute phase, less prevalent to diabetes development, however, their intes- when TJs are open, this suggests zonulin as the key mediator tinal permeability was not altered suggesting that it is an event in the pathology of coeliac disease. Further, an increase in preceding the disease (Meddings et al., 1999). Similarly, in a expression as a result of TJ displacement may provide study carried out on children suffering from coeliac disease, increased zonulin presentation to immune cells in the sub- increased intestinal permeability and abnormal TJ structure mucosa thus resulting in an autoimmune response. To verify was present and not entirely reversible following gluten- this hypothesis, ZOT-based ELISA was used to measure free diet implementation. These results suggest either irrevers- zonulin antibody levels in the blood of coeliac disease suf- ible damage is made to the intestinal epithelium during coeliac ferers against a control group. While anti-zonulin IgG levels disease, or alteration in TJ complexes before disease onset were similar in patients with coeliac disease and in controls, thus eliminating the disease will not eliminate the prior symp- anti-zonulin IgA levels were elevated in 21% of coeliac dis- toms (Schulzke et al., 1998). To determine the correct explan- eased patients during the acute phase of the disease. In remis- ation however, one would need to examine individuals prior sion, individuals’ anti-zonulin IgA levels and intestinal to the onset of the disease, however identifying such indivi- permeability returned to normal after 3–6 months on a duals proves challenging. gluten-free diet, further evidencing zonulin as the main con- Epithelial dysfunction prior to and during coeliac disease is tributor to disrupted barrier function. restricted to the jejunum and distal ileum, while the colon Irish setter dogs were used in an experiment to provide evi- permeability remains unperturbed (where the presence of dence for increased intestinal permeability participating in the microflora and byproducts could be detrimental upon com- aetiology of coeliac disease (Hall and Batt, 1991). The litters promised epithelium). This coincides with the regional distri- were bred from gluten-sensitive enteropathy suffering dogs. bution of ZOT receptors, with the ZOT-induced F-actin The experiment compared litters predisposed to disease redistribution in the villi, and with the detection of immuno- reared on a gluten-free diet and on a normal diet, compared fluorescent particles within the gastrointestinal tract studied to a control group of healthy dogs on a normal diet. in rabbit intestine (Fasano et al., 1997). Similar results have Permeability was found to have risen in the litters on a gluten- been described in human intestine (Uzzau et al., 2001). free diet with unaffected intestinal integrity. In litters reared Decreasing ZOT receptors along the villus-crypt axis sug- on a normal diet, increased intestinal permeability was noted gests upregulation during enterocyte differentiation of ZOT compared to that in gluten-free diet litters after just 4 months receptors. and elicited villus degeneration, intraepithelial lymphocyte By chance, Fasano (2011) uncovered the key to unlocking infiltration and reduced ALP activity. The findings support the means of oral delivery of large-molecule drugs across the the hypothesis that intestinal hyperpermeability is a primary intestinal epithelial layer. Earlier attempts to open up TJs defect involved in eliciting gluten-sensitive enteropathy in between epithelial cells failed due to the complete and irre- Irish setter dogs. It is important to note however, that the versible destruction of the TJ, differing from zonulin’s valu- gluten-free diet may have contained other elements potentiat- able reversible displacing effect. In early studies, Fasano ing sensitivity. Carrageenan is an additive often found in dog showed ZOT’s ability to induce a 10-fold increase in insulin food and is found to cause gastrointestinal inflammation, absorption and six-fold increase in absorption of immunoglo- increased susceptibility to intestinal lesions, ulcerations and bulins in rabbit small intestine (Fasano and Uzzau, 1997). tumours (Tobacman, 2001). For more pertinent results, the Diabetic rats were also studied, where ZOT administration litters could be reared on an elemental diet, a liquid diet in demonstrated a greater decreased in blood glucose levels com- which only the basic nutrients the body requires are ingested, pared to those derived following insulin injections. This opens in order to eliminate other hyperpermeability potentiating ele- up the possibility of replacing painful insulin injections with ments. Thus increased intestinal permeability is most likely oral pill ingestion. The oral delivery of drugs such as taxol not the primary defect, but rather, secondary to the presence and growth hormone in conjunction with zonulin could, for of a dietary elements potentiating increased intestinal perme- example, be considered for treating cancer. The discovery ability (Hall and Batt, 1991). Regardless, the experiment may also change the lives of many autoimmune disease suf- strongly suggests hyperpermeability precedes disease. ferers. Knowing that increased permeability of TJs predis- The opening of TJ in coeliac disease if left untreated has poses individuals to autoimmune diseases suggests that with been reported to increase chances of other autoimmune dis- appropriate permeability reinstated, likelihood of disease ease development such as type I diabetes mellitus, development is reduced. ............................................................................................... .................................................................. 4 Bioscience Horizons � Volume 10 2017 Review article ............................................................................................... .................................................................. The discovery of zonulin, a potential tool for drug and pep- A study in humans revealed early introduction of gluten- tide transport through the intestinal epithelium, is tremen- containing grains to infants increased risk of diabetes (Norris dously exciting. ZOT displays many useful properties making et al., 2003). This study looked at 900 infants with increased it a suitable candidate for the job: it is non-cytotoxic; it does T1D risk, defined by exhibiting the T1D-susceptible HLA not irreversibly destructs the epithelium, but rather, displaces gene or by having a first-degree relative with T1D. The infants TJs in a reversible manner; it interacts with a region-specific were tested at 9, 15 and 24 months and annually thereafter receptor (specific to jejunum and distal ileum); it is not effect- for insulin autoantibodies, glutamate decarboxylase (GAD) ive in the colon where it could potentiate harm via intestinal autoantibodies and islet antigen-2 (IA-2) autoantibodies by barrier disruption; it does not induce acute systemic side means of a blood test. Testing positive in at least one of these effects; and it acts relatively quickly (increased permeability autoantibodies or having diabetes itself meant testing positive detected 20 min after oral administration). for islet autoimmunity. The results showed that infants exposed to gluten between ages 0–3 months and 7+ months resulted in an elevated risk of T1D development while expos- Type I diabetes mellitus ure to the cereal between 4 and 6 months resulted in a reduced or delayed risk of T1D development. Curiously, this may indi- Type 1 diabetes mellitus (T1D) is defined by hyperglycaemia cate a window of exposure to gluten in infancy outside of instigated by an autoimmune destruction of insulin- which islet autoimmunity and T1D risk in susceptible infants producing β-cells in the pancreas. Various genes have been is increased. The study also found that if gluten-containing identified to be involved in T1D susceptibility. The main two grains were introduced while the infant was still breastfeed- are the HLA gene region IDDM1, located on chromosome ing, the risk of islet autoimmunity and T1D was reduced, 6p21 and accounting for 42% of genetic susceptibility, and independent of the window of exposure previously the insulin gene region IDDM2, on chromosome 11p account- calculated. ing 10% of genetic susceptibility. Other known chromosomal Administration of antibiotics such as doxycycline, fusidic locations with undetermined susceptibility genes have been acid, colistin and Bactrim to BBDP rats and NOD mice after described (Kelly et al., 2003). Additionally, polymorphisms in weaning leads to the prevention of T1D development. MYO9B, a gene involved in intestinal permeability have also Although the mechanism is not entirely understood, micro- shown interrelation with T1D development (Santiago et al., biota dysbiosis affects the development of T1D in both animal 2008). models. This is further supported by a study administrating Subjected to particular environmental triggers, genetically probiotics to NOD mice which was found to induce IL-10 predisposed individuals may develop T1D. Such triggers have production, an immunoregulator and anti-inflammatory cyto- yet to be identified, and would allow for prevention and treat- kine, and to prevent T1D development (Calcinaro et al., ment of the disease. Gliadin is believed to be a T1D trigger, its 2005). Another study revealed neonatal administration of an mechanism similar to that in coeliac disease. Non obese dia- immunodominant epitope of T1D, peptide p277, proved to betic (NOD) mice and BBDP rats have been used to demon- potentiate the protective effect of a hydrolysed casein diet, strate that gliadin is a dietary trigger for T1D showing reducing the incidence of T1D by 64% or delaying its devel- increased intestinal permeability following wheat administra- opment (Brugman et al., 2004). tion. Rearing NOD mice and BBDP rats on a gluten-free diet The jejunum of T1D sufferers have been found to display resulted in delayed and reduced T1D development. The study increased IFNγ and TNF-α,inflammatory response factors also showed that time of exposure to the trigger is important: leading to pancreatic damage, their amounts correlating with delayed gluten exposure by prolonging breastfeeding led to the severity of the disease (Westerholm-Ormio et al., 2003). reduced T1D development in BBDP rats (Visser et al., 2003). Significantly, a Th1 bias has been found in the mediastinal The experiment investigated the relationship between dur- lymph node (MLN), a gut-associated secondary lymphoid ation of exclusive breastfeeding and the onset of T1D. organ where APCs present soluble dietary antigens to naïve T Normal rat breastfeeding duration is 21 days, in this experi- cells, of T1D individuals (Chakir et al., 2005). In healthy indi- ment however the lactation period was prolonged to 31 and viduals, the gut mucosa is a Th2 predominant environment 40 days. In BBDP rats on a 31 day breastfeeding diet, reduced which allows for normal immunosuppressive response to glu- onset of T1D was noted compared to that of the 21 day ten. In T1D rats, however, a Th1 bias is apparent, resulting breastfeeding control group. Surprisingly, rats on a 40 day from a Th2-specific Gata3 deficit causing an imbalance in breastfeeding diet showed unchanged risk of T1D and were just Th1/Th2 differentiation. Elevated Th1 levels results in as prone to T1D as the control rats. These rats were underweight increased IFNγ and TNF-α production leading to pancreatic andexhibited reducedgrowthratecharacteristic of malnutrition, islet β-cell damage. Chakir and his team also found gluten- known to reduce β-cell function in later-life. Poor functioning of specific CD4+ T cells in the MLN of early-stage diabetic rats. β-cells impairs the insulin-secreting capacity of the pancreas. The Low frequencies of the cells were found in the spleen, suggest- study draws the conclusion that while increased exclusive breast- ing activation of CD4+ T cell in the gut. This experiment links feeding period may reduce T1D risk, excessive prolongation of the gut immune system and T1D. this period may annul the positive effect. ............................................................................................... .................................................................. 5 Review article Bioscience Horizons � Volume 10 2017 ............................................................................................... .................................................................. Bosi et al. (2006) showed increased intestinal permeability gene expression in intestinal mucosal cells following exposure to precede T1D. He studied 81 subjects with islet autoimmun- to gliadin in T1D individuals demonstrating intestinal T-cell ity at varying stages (preclinical, newly-onset and long-term activation by gliadin (Auricchio et al., 2004). established diabetes) and 40 control subjects and submitted In conclusion, individuals with T1D are either genetically them to the lactulose–mannitol test. Orally administered, the predisposed, have been subjected to environmental triggers or two sugars were measured in urine to extrapolate intestinal experienced gut microbiota dysbiosis and display increased permeability. Disaccharide lactulose, a larger molecule, intestinal permeability, heightened immune activation (e.g. crosses the intestinal epithelium intracellularly via TJs while inflammation), histological changes and elevated plasma monosaccharide mannitol, a smaller molecule, is transported zonulin. In contrast to coeliac disease with the sole trigger of across the epithelium transcellularly through water-filled gliadin, T1D is believed to have several environmental trig- pores on the cell membrane. Altered intestinal anatomy or gers. Bovine insulin, in cow milk, has been shown to elicit functional integrity would result in facilitated passage of one similar reactions to gliadin resulting in T1D development. or both molecules. Intestinal permeability to lactulose was Bovine insulin administration in infants may sensitize intes- shown to be elevated in all islet autoimmunity individuals tinal T-cells later partaking in pancreatic insulin-producing β- indicating barrier damage while integral surface mucosa cell destruction, an autoimmune response. Evidently further remaining intact shown by unchanged permeability to manni- research is necessary wholly to understand the role of the gut tol compared to controls, thus increasing the lactulose:mani- and its microbiota in T1D. However, it is understood that tol ratio. This study supports the hypothesis that increased microbiota dysbiosis causes upregulation of gut and systemic intestinal permeability precedes T1D. inflammation, due to endotoxins from pathogenic bacteria or The morphological aspect of the intestinal mucosa in non- to pro-inflammatory cytokines synthesized by immune cells, coeliac T1D individuals was studied by Secondulfo et al. and causes T1D as well as disrupting gut–brain axis commu- (2004) to investigate reasons for increased permeability. Four nication potentially leading to neuroinflammation and cogni- parameters of the mucosal ultra-structural were evaluated: tive dysfunction (e.g. Alzheimer’s) (Bhattacharjee and Lukiw, microvilli height, microvilli thickness, distance between 2013). Identifying the particular environmental triggers and microvilli, and TJ thickness. Reduced density, displacement improving investigative methods will greatly help evaluate the and abnormal (short and thin) formation of microvilli were intestinal epithelium and hopefully provide important insights found on the apical membrane enterocytes, many of which towards better understanding the pathogenic process of the contained large vacuoles, engorged mitochondria and a sur- disease and consequentially potentiating prevention and plus of lysosomes. Enterocyte intercellular spaces were treatment. enlarged and TJ domains were displaced and thickened. The disturbed intestinal epithelium function in non-coeliac T1D Discussion individuals was associated with altered mucosal morphology suggesting loss of function of the intestinal barrier leading to As evidenced, autoimmune disorders develop over time and pathogenesis. can be preclinically detected. Due to their ambiguous symp- A wide collection of studies demonstrate synergism toms (fatigue, muscle or joint ache, general malaise), correct between microbiota dysbiosis, increased intestinal permeabil- diagnosis of autoimmune disorders can prove difficult. Many ity, altered mucosal immunity and pathogenesis of T1D. autoimmune disease sufferers wait until symptoms aggravate Kuitunen et al. (2002) found an increased lactulose and man- and the disease begins to take its toll before seeking medical nitol absorption in T1D sufferers thought to be due to altered advice, at which stage disease reversal may no longer be intestinal permeability (Kuitunen et al., 2002, Secondulfo possible. et al., 2004, Sapone et al., 2006). Increase in absorption of The majority of autoimmune disorders are not well under- the two sugars was also found to be the case for β-cell auto- stood. Identification of the triad of triggers—genes, environ- immune individuals suggesting altered intestinal permeability mental factors and microbiotia dysbiosis—as well as prior to the onset of T1D (Bosi et al., 2006). Histological improvements of investigative techniques will allow better changes in height and thickness of intestinal villi as well as understanding of the pathological processes involved and per- their spacing and TJ assembly was altered in T1D individuals mit treatment development. Understanding such functioning signifying the presence of mucosal injury (Secondulfo et al., will allow prevention of disordered intercellular communica- 2004). Structural changes in TJ complexes in T1D were tion and digestive disorders leading to autoimmune diseases, understood from recordings of elevated plasma zonulin levels tissue inflammation, harmful adaptations and metastasis. in individuals with T1D (Sapone et al., 2006). Several experi- Remarkably, the same system which can cause such damage ments have investigated intestinal biopsies in children with could also be the key to curing hundreds of diseases through T1D and found elevated interleukin and interferon levels tissue specific drug delivery. indicating intestinal inflammation (Savilahti et al., 1999, Westerholm-Ormio et al., 2003). Auricchio and his team Fasano unknowingly uncovered an innovative strategy for + + found heightened CD3 and CD25 cells and increased HLA the delivery of drugs, macromolecules and vaccines across the ............................................................................................... .................................................................. 6 Bioscience Horizons � Volume 10 2017 Review article ............................................................................................... .................................................................. body’s various epithelia in an innocuous and minimally Acknowledgements inflammatory manner. Interestingly, not only intestinal but extra-intestinal epithelia such as tracheobronchial and renal I wish to express my sincere gratitude to Dr Michael Lucas, my tubule, as well as the vascular endothelium including the supervisor, for his guidance, enthusiasm and invaluable advice blood brain barrier may be targeted. on this subject. I would also like to thank my friends and family, for their encouragement to complete this dissertation. Zonulin analogues, such as AT-1002, have been developed and promote tissue penetration of macromolecules across a wide variety of surfaces while preserving membrane integrity thus altering permeability in a non-destructive, transient and References reversible manner. Multiple delivery routes mean facilitated administration of the vaccine or drug increasing patient com- Arrieta, M. C., Bistritz, L. and Meddings, J. B. (2006) Alterations in intes- pliance and appeal to practitioners. AT-1002 has been tinal permeability, Gut, 55 (10), 1512–1520. demonstrated on nasal epithelia, intestinal epithelia and cul- Auricchio, R., Paparo, F., Maglio, M. et al. (2004) In vitro-deranged intes- tured brain endothelial cells. It can be used to transport PEG tinal immune response to gliadin in type 1 diabetes, Diabetes,53 or insulin across the intestinal epithelium for treatment of (7), 1680–1683. constipated or diabetic individuals (Fasano and Uzzau, 1997, Song, Fasano and Eddington, 2008). AT-1002 can also Bhattacharjee, S. and Lukiw, W. J. (2013) Alzheimer’s disease and the induce rapid and reversible permeability of the blood brain microbiome’, Frontiers in Cellular Neuroscience, 7, 153. barrier and thus increase absorption of doxorubicin and Bosi, E., Molteni, L., Radaelli, M. G. et al. (2006) Increased intestinal per- paclitaxel for cancer treatment (Karyekar et al., 2003). The meability precedes clinical onset of type 1 diabetes, Diabetologia, analogue also instigates protective immune responses by adju- 49 (12), 2824–2827. vant mucosal antigen delivery when administered nasally or rectally (e.g. ovalbumin and tetanus toxoid (Marinaro et al., Brugman, S., Klatter, F. A., Visser, J. et al. (2004) Neonatal oral adminis- 2003)). Cyclosporine transported with AT-1002 is used in the tration of DiaPep277, combined with hydrolysed casein diet, pro- prevention of transplant rejection, Crohn’s disease and tects against Type 1 diabetes in BB-DP rats. An experimental study, rheumatoid arthritis. It has also been experimentally tested to Diabetologia, 47 (7), 1331–1333. treat aids (AT-1002 taken along with ritonavir and saquina- vir) and viruses such as varicella, herpes genitalis and zoster Calcinaro, F., Dionisi, S., Marinaro, M. et al. (2005) Oral probiotic admin- (with acyclovir) (Salama et al., 2005). istration induces interleukin-10 production and prevents spontan- eous autoimmune diabetes in the non-obese diabetic mouse, To summarize, zonulin agonists can be used to reversibly Diabetologia, 48 (8), 1565–1575. increase paracellular transport of drug delivery with less tox- icity than previous absorptive enhancers, while antagonists Campbell, A. W. (2012) Organic vs conventional, Alternative Therapies in can be used to prevent increased absorption of pathogenic Health and Medicine, 18 (6), 8–9. molecules or allergens. Zonulin agonists and antagonists have Campbell, A. W. (2014) Autoimmunity and the gut, Autoimmune the potential for significant and effective use in pharmacology Disease, 2014, 152428. not only in autoimmune disorders but in all diseases. Chakir, H., Lefebvre, D. E., Wang, H. et al. (2005) Wheat protein-induced In conclusion, we know significantly more today than we proinflammatory T helper 1 bias in mesenteric lymph nodes of did 5 years ago and it is to be hoped that we may be able to young diabetes-prone rats, Diabetologia, 48 (8), 1576–1584. say the same in 5 years’ time. The non-destructive opening of tight junctions means ‘open sesame’ to new therapies. Large Fasano, A. (1999) Cellular microbiology: can we learn cell physiology long-term studies, however, are required to determine poten- from microorganisms? The American Journal of Physiology, 276 (4 Pt tial adverse effects of possible new therapeutic approaches. So 1), C765–C776. far, the results look promising. Since it is now known that Fasano, A. (2008) Physiological, pathological, and therapeutic implications hyperpermeability precedes disease, a new and innovative of zonulin-mediated intestinal barrier modulation: living life on the approach to treatment, arresting the interplay between genes edge of the wall, The American Journal of Pathology, 173 (5), 1243–1252. and environmental factors through intestinal barrier function re-establishment, can be used in prevention or treatment of Fasano, A. (2011) Zonulin and its regulation of intestinal barrier func- autoimmune disorders. tion: the biological door to inflammation, autoimmunity, and can- cer, Physiological Reviews, 91 (1), 151–175. Author biography Fasano, A. (2012a) Leaky gut and autoimmune diseases, Clinical Reviews in Allergy & Immunology, 42 (1), 71–78. Graduated in 2016 with a First Class Honours in Physiology Fasano, A. (2012b) Zonulin, regulation of tight junctions, and auto- (BSc) from the University of Glasgow. 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