Pivotal Role of Inflammation in Celiac Disease

Pivotal Role of Inflammation in Celiac Disease: Comparison

Please note this is a comparison between Version 1 by Maria Vittoria Barone and Version 2 by Conner Chen.

Celiac disease (CD) is an immune mediate disease characterised by gluten dependent T-cell mediated activation, autoimmunity and derangement of the intestinal mucosa in a specific genetic background. Although the activation of the T-cells has been studied in dept, the central question remains still unanswered, namely, why a pro-inflammatory T cell response to gluten is generated instead of a regulatory response, which normally promotes oral tolerance to dietary protein antigens.

Recent Tliterature demonstrated that there is an inflamed environment in CD intestine, enriched in cytokines, such as IL-15, or type I interferons, in which T cells tend to acquire a pro- inflammatory phenotype. The factors that create a pro-inflammatory environment in the CD intestine, leading to an expansion of gliadin-specific T cells in genetically susceptible individuals and further shifting them towards a pro-inflammatory phenotype, remain to be identified. The recent literature is starting to address this question. From these studies it appears clearly, that subtle alterations on the CD cells are present also before the introduction of gluten. Gluten exacerbates these constitutive alterations, by increasing the same markers already altered before the gluten introduction, both in vitro and in vivo.

All these new observations add this disease “tout court” to the big family of increasing chronic inflammatory diseases where nutrients can have pro-inflammatory or anti-inflammatory effects, directly or indirectly mediated by the intestinal microbiota, where the intestine functions as a cross road for the control of the inflammation both local and at distance.

celiac disease
gluten
inflammation
microbiota

1. Celiac Disease as an Inflammatory Chronic Disease

The recent literature describes in celiac disease (CD) a meeting of several different factors such as cellular vulnerability, pro-inflammatory effects of gluten and other wheat proteins, Western diet, and other environmental triggers such as viruses that prepare and/or amplify the T cell-mediated response to gluten. The factors that create a pro-inflammatory environment in the CD intestines, leading to an expansion of gliadin-specific T cells in genetically susceptible individuals and further shifting them toward a pro-inflammatory phenotype, could have multiple origins: the pro-inflammatory environment (exogenous stimuli), such as diet, viruses and other pro-inflammatory factors; and the constitutive cellular alterations (endogenous predisposition) that by themselves induce and/or render the cells more sensitive to pro-inflammatory stimuli. All these factors, both exogenous and endogenous, can contribute to the generation of “sterile” inflammation in CD (Figure 1).

Figure 1. Both exogenous and endogenous factors can generate low-grade chronic inflammation in CD, initiating a series of events that will eventually induce an intestinal lesion.

2. Endogenous Alterations in CD Independent of Gluten

Endogenous alterations, defining a celiac cellular phenotype, have been described in different tissues and cells from CD patients, including intestinal organoids ^[1][17]. Gluten exacerbates these constitutive alterations by increasing the same markers already altered in the absence of gluten, both in vitro and in vivo. This phenotype confers the vulnerability to the CD cells to several different triggers that have effects on different pathways, including innate immunity activation. Many of these constitutive alterations are now regarded as biomarkers of clinical relevance, as they can be used to intervene in the “at risk” population before the onset of the disease ^[1][2][17,18].

The recent literature is starting to address the question of endogenous alterations, independent of gluten, in CD by in vivo studies and in cellular models. From these studies, it clearly appears that subtle alterations of the CD cells are also present in the absence of gluten as well as in the absence of T cell activation.

ThWe dere are many scribe the literatures including the concerning these points by starting with population studies, such as patients at risk of CD, CD patients with GFD–CD (gluten-free diet–celiac disease) and in vitro studies on biopsies, intestinal organoids and CD cells from compartments different from the intestines (Table 1).

Table 1. Endogenous, constitutive alterations of several pathways have been described in different cellular models of CD. Most of these endogenous alterations can predispose one to inflammation. Some of these constitutive alterations can be regarded as biomarkers of CD. CD: celiac disease; Wnt: Wingless and Int 1; NFkB: nuclear factor kappa-light-chain-enhancer of activated B cells; EGFR: epithelial growth factor receptor; ERK: extracellular signal-regulated kinases; ECM: extracellular matrix; pNFkB: phosphorylated (active) form of NFkB; pERK: phosphorylated (active) form of ERK; IL1 beta: interleukin beta 1; IL6: interleukin 6; LPP: lipoma-preferred partner; IL15: interleukin 15; IL15R alpha: IL15 receptor alpha; tTg: tissue transglutaminase.

Endogenous Factors
Models Investigated	Pathways Described
Children at risk of CD (before gluten introduction)	(a) Lipid profiles in the blood ^[3]^[4][19,20] (b) Impaired growth of children ^[5][21] (c) Inflammation markers in the blood ^[6]^[7][22,23] (d) Gut microbiota changes ^[8][24]
GFD–CD biopsies	(a) Altered expression of genes related to: brush border assembly, development, transport cell cycle ^[9][25] (b) Wnt signaling alterations ^[9][25] (c) Epithelial inflammation, crypts hyperplasia, low grade inflammation in the serum ^[10]^[11][26,27] (d) NFkB pathway alterations ^[12]^[13]^[14]^[15][28,29,30,31] (e) Innate immunity activation ^[16]^[17]^[18]^[19]^[20]^[21]^[22][32,33,34,35,36,37,38] (f) EGFR/ERK mediated proliferation in the crypts ^[23]^[24]^[25]^[26][39,40,41,42] (g) Tight junctions and permeability alterations ^[27]^[28]^[29]^[30]^[31]^[32][6,43,44,45,46,47] (h) Impairment of vesicular trafficking ^[23][39]
Intestinal organoids derived from CD patients	(a) Increased staminality and permeability ^[33][48] (b) Increased inflammasome and innate immunity genes ^[33][48] (c) ECM genes decreased ^[34][49] (d) Increased pNFkB, pERK, IL beta, IL 6 ^[1][17]
Nonintestinal cells from GFD–CD patients	(a) In fibroblasts: Cell shape ^[23]^[26][39,42] Increased inflammation and innate immunity markers ^[23][39] Focal adhesions markers and LPP altered expression ^[35][50] Increased IL15 and IL15R alpha expression ^[23][39] Differential features of tTg ^[36][51] In dendritic cells: Cell shape alterations ^[37][52]

2.1. Patients at Risk of CD: What Happens before the Disease?

Patients at risk of CD are those subjects that have a genetic risk defined as the presence of HLA (human leukocyte antigen) DQ2 or 8 and are first-degree relatives (sons or daughters, sisters or brothers) of a CD patient. These subjects were followed from birth in prospective studies for several years. Studies on this population also had the aim to find predictive markers of the disease years before its onset ^[38][53] (Table 1).

In a recent study ^[3][19], infants at risk for CD were followed up to 8 years to monitor the onset of CD. In infants that developed CD, the serum phospholipid profile was altered at 4 months, long before any exposure to gliadin or any production of anti-tTg (tissue transglutaminase) antibodies, compared to their controls matched for the same genetic profile that during the 8 years of observation did not become celiac patients. The phospholipid signature was dramatically different in infants who developed CD when compared to that of infants at risk not yet developing CD. A specific phospholipid profile was able to discriminate infants who eventually developed celiac disease years before antibodies or clinical symptoms appeared. This lipid profile appears to be constitutive since it is constant over time and is already present at 4 months of age ^[3][19]. Other groups have obtained similar results in CD ^[4][20]. Most of the lipids found altered in theirs study perform an important structural role in the plasma membrane, contributing not only to membrane integrity but also to intracellular vesicle functions. Recent studies have shown that vesicle trafficking might be a crucial key in understanding the handling of gliadin peptides in celiac patients ^[23][39][40][39,54,55] (Table 1). Moreover, lipids not only have structural functions in the composition of membranes and organelles, they also have crucial signaling roles over and above their nutritional function. Therefore, lipid metabolism and its unbalance can contribute to acute and chronic inflammation in many different conditions and ways ^[41][56]. Previous lipid omics studies found that dysregulated lipid metabolism precedes islet autoimmunity and overt disease in children who later progressed to T1D, indicating that alterations in lipid metabolism can have a central role in autoimmune diseases ^[42][43][57,58].

The impaired growth of children, as assessed at the time of CD diagnosis, is a common manifestation of CD, although in CD patients on a gluten-free diet, there is no growth impairment. In the at-risk population, alteration in growth has been described before gluten introduction or before the appearance of any signs of the disease (specific antibodies or symptoms). The growth rate of at-risk subjects that developed CD and the controls were normal at birth, but early on, at 4 months, differences were present between the two groups ^[5][21].

In these same at-risk subjects, there was low-grade inflammation with an increase in pro-inflammatory cytokines in the serum before gluten introduction. At 4 months before gluten introduction, the levels of a certain set of proinflammatory cytokines were higher in the serum of predisposed infants who developed celiac disease compared to controls. These results suggest the presence of an “inflamed” background before the exposition to gluten. The genetic profile of infants who develop CD shows a number of differential SNPs (single-nucleotide polymorphisms) in the gene associated with CD compared to those who do not develop the disease, up to 6 years ^[6][7][22,23] (Table 1).

The possible hypothesis to explain these early alterations in CD patients is that a genetic predisposition could modulate the serum cytokine pattern before gluten introduction, which might create a dangerous background for the introduction of gluten during the following years.

Finally, a recent study suggests shifts in the early trajectory of the gut microbiota along with changes in immune markers in infants before weaning who later develop CD, indicating deviations of the normal microbiota maturation process before CD development ^[8][24] (Table 1).

In conclusion, there are several important implications. In CD, some constitutive alterations in metabolic, inflammatory and structural pathways are present before the onset of the disease, before gluten introduction in the diet, and consequently, before the gluten-induced autoimmune response. Thus, constitutive alterations may contribute to creating a chronic inflammatory condition that precedes and prepares the T cell-mediated response.

2.2. GFD–CD Patient Biopsies, Lessons from In Vivo and In Vitro Studies

Celiac patients after diagnosis have to initiate a strict diet without gluten. These patients are defined as gluten-free diet–celiac disease patients (GFD–CD). The dietary restrictions have to be followed for life. GFD–CD patients do not eat gluten; thus, the main cause of the disease is not present, but some alterations mainly at the intestinal level can still be found.

GFD–CD Patients before and after Gluten Challenge In Vivo

New observations have been obtained and published challenging gluten in vivo in GFD–CD patients by analyzing before and after gluten introduction on several different pathways such as proliferation, inflammation and differentiation at the intestinal level ^[9][10][11][25,26,27]. Dotsenko et al., demonstrated that the intestinal morphology in patients on a strict GFD was similar to that measured in control subjects ^[9][25]. However, gene transcription showed that the GFD–CD group differed significantly from the control group, showing a substantial number of differentially expressed genes. Gene Ontology and Reactome pathway analyses revealed that patients on a GFD presented altered expression of genes with functions such as brush border assembly, developmental processes, transport of small molecules, and FOXO (Forkhead box-O)-mediated transcription of cell cycle genes ^[9][25]. Moreover, the Wnt (Wingless and Int 1) pathway has been found altered in GFD–CD patients with respect to controls, suggesting that the alterations of a pathway nodal to intestinal differentiation and homeostasis is also present in the absence of gluten ^[9][25]. In the gastrointestinal tract, Wnt signaling activation drives homeostasis and damage-induced repair. Wnt signaling also has a key role during stem cell-driven intestinal homeostasis, regeneration, ageing and cancer ^[44][59]. After being gluten challenged, the Wnt pathway was further altered, as tested by RNA sec, in intestinal biopsies of patients on a GFD with respect to controls ^[9][25]. All together, these data show that even on a strict GFD, patients reveal patterns of ongoing disease and that gluten acts on the same pathways already altered. Another recent paper by Stamnaes J. et al. ^[10][11][26,27] shows by proteomics analysis that before being challenged in GFD–CD patients, epithelial inflammation is already in the intestines, with changes indicative of minor crypt hyperplasia and low-grade inflammation in the serum. After challenge with gluten, an increase in the same proteins, already altered at base line in GFD–CD responders, was found. Despite clinical and histological remission, celiac disease patients that develop a mucosal response after 14 days of gluten challenge have already at baseline altered protein compositions of their gut tissue with signs of ongoing inflammation. In conclusion, these observations indicate that in GFD–CD patients, several different pathways such as proliferation, inflammation and differentiation are altered at the intestinal level and that they can be further altered by gliadin challenge.

GFD–CD Patient Biopsies

Several papers have been published studying the biopsies of GFD–CD patients. In these papers, several pathways have been found altered by different techniques. Most of the papers analyze the whole biopsies, as in the case of genetic or expression studies, while in some others, only the epithelium are evaluated. The following listed are the most significant literature contributions that we discuss, clustered by the pathways found altered.

A.

Inflammation

Several different genetic ^[12][28] and expression ^[13][14][15][29,30,31] studies indicate that the NFkB (nuclear factor kappa-light-chain-enhancer of activated B cells) pathway is altered in CD. It is widely accepted that NFkB is a key regulator of inducible gene expression in the immune system. It has also been shown that NFkB is a mediator of IL15, a major player of innate immunity in CD. Both innate and adaptive immune responses, as well as the development and maintenance of the cells and organs as well as homeostasis that comprise the immune system are, at multiple stages, under the control of the NFkB family of transcription factors. Moreover, NFkB is responsible for the transcription of genes encoding a number of pro-inflammatory cytokines and chemokines ^[45][60]. Fernandez-Jimenez et al. ^[13][29] showed that the expression of 93 NFkB genes measured by RT-PCR was altered in a set of uncultured active and treated CD patients with respect to control biopsies, and in cultured biopsies from CD at different stages of the disease (GCD–CD and GFD–CD) challenged with gliadin. These results showed that genes constitutively upregulated in GFD–CD patients belonged to the essential core of the pathway and had crucial, regulatory and central roles in the NFkB signaling system, whereas genes that were overexpressed only in active CD appeared to be more peripheral and included mostly NFkB-inducible interleukins, adhesion molecules and receptors. Moreover, gluten challenge on GFD–CD biopsies increased the NFkB pathway ^[13][14][15][29,30,31]. The lncRNA13 (long non coding RNA 13) levels are significantly decreased in biopsy samples from the small intestines of CD patients, leading to poor control of the repressed pro-inflammatory genes, and possibly contributing to underlying chronic inflammation ^[14][15][30,31].

B.

Innate immunity pathways

Stress signals are expressed by the enterocytes of patients with celiac disease ^[16][32]. In the duodenal mucosa of children with untreated CD as well as in children with treated CD, compared with those in controls, elevated HSP72 (Heat shock 70 kDa protein 1) mRNA expression and higher protein levels were found ^[17][33]. Moreover, innate immunity has been found activated in GFD–CD patients. The most studied cytokines in GFD–CD biopsies are: IL15 (interleukin 15)/IL15R (IL15 receptor) and INF-α (Interferon α) ^[18][19][20][34,35,36]. IL15 is a cytokine whose role is essential to the control of immune homeostasis; its expression is tightly regulated at the translational, transcriptional, and intracellular trafficking levels ^[21][37]. A high number of CD patients on a GFD–CD maintain high levels of IL-15 expression in the epithelium ^[18][34]. IL15R alpha mRNA expression and proteins were increased in CD patients, as compared with non-CD controls. Moreover, CD patients show increased sensitivity to IL-15 stimuli to produce both nitrites and IFN (Interferon) gamma ^[19][35]. A mouse model bearing the predisposing HLA-DQ8 molecule and that reproduces the overexpression of IL-15 both in the gut epithelium and the lamina propria (LP) features characteristics of active CD, having clarified the role of IL 15 in the pathogenesis of CD. These mice upon gluten load develop villous atrophy (VA) ^[22][38]. The link between infections and CD has been established clinically by several large-scale, population-based cohort studies ^[46][61]. Another player of the innate immunity response to gliadin in CD seems to be IFN-α and its downstream effector MxA (Myxovirus resistance protein A) ^[18][34], both involved in the Toll-like receptor response to external agents, such as viral infections. Higher levels of expression of MxA and IFN-α were found in GFD–CD biopsies with respect to controls before and after treatment with gliadin in GFD–CD biopsies ^[20][36].

C.

Enterocyte proliferation and differentiation

Damage to the intestinal mucosa in CD is mediated both by inflammation due to the adaptive and innate immune response to gliadin and by proliferation of crypt enterocytes as an early alteration of CD mucosa causing crypt hyperplasia ^[47][48][49][62,63,64]. The celiac intestines are characterized by an inversion of the differentiation/proliferation program of the tissue, with a reduction in the differentiated compartment, up to complete villous atrophy, and an increase in the proliferative compartment with crypt hyperplasia ^[24][25][40,41]. In CD biopsies both from GCD– and GFD–CD patients, increased activity of the EGFR (epidermal growth factor receptor)/EGF (epidermal growth factor) system and the downstream signaling molecule ERK (extracellular signal-regulated kinases) has been described ^[23][24][26][39,40,42] together with the enhancement of crypt enterocyte proliferation dependent on EGFR/ERK activation ^[23][24][26][39,40,42]. Gluten and gliadin peptide p31-43 can increase proliferation in GFD–CD patient biopsies both in vitro ^[39][50][54,65] and after gluten challenge in vivo ^[51][66].

D.

Structural alterations

The intestinal epithelial tight junction (TJ) barrier controls the permeation of the intestinal lumen contents into the intestinal tissue and consequently into systemic circulation. A defective intestinal TJ barrier has been implicated as an important pathogenic factor in inflammatory diseases of the gut, including Crohn’s disease, ulcerative colitis, necrotizing enterocolitis, and celiac disease. CD-altered intestinal permeability may facilitate the entry of gluten or its fractions into the lamina propria where it can cause a series of immunological events. Permeability of the intestinal mucosa has been tested by several different methods in vivo and in vitro ^{[28][29][30][31][52][53]}[43,44,45,46,67,68] in CD patients on a GFD. Persistently increased mucosal permeability to certain probes (molecular weight less than 1500 Daltons) has been shown in patients with celiac disease on a GFD with normal intestinal histology ^[29][44]. Patients with both celiac disease and dermatitis herpetiformis show persistent increase in intestinal permeability, which suggests a common pathogenetic mechanism for both disorders ^[28][43]. Moreover, barrier dysfunction and structural alterations found in acute CD were only partially recovered in GFD–CD patients, suggesting a level of “minimal damage” ^[30][31][45,46] in CD patients on a GFD. Finally, the impact of altered intestinal permeability may have a role in the secondary autoimmune phenomena as well as the extraintestinal manifestations of CD. CD patient’s antibodies can have biological effects on intestinal and other cells. CD patients may present autoantibodies directed against extraintestinal antigens such as antineuronal and antiganglioside antibodies (more prevalent in patients with neurological disorders) ^[52][53][54][67,68,69]. CD patients on a gluten-containing diet and severe mucosal damage (villous atrophy) frequently present anti-actin IgA antibodies that display high specificity for CD and disappear after GFD, as previously reported ^[55][70]. In addition, a link between atopy and CD has been suggested by an Italian study ^[56][71]. Alterations at the genetic and expression levels have been found for proteins involved in the regulation of permeability and tight junctions in CD ^[32][57][58][47,72,73], suggesting that at least partially, this alteration of permeability can be attributed to genetic defects. A possible alternative or additive mechanism to genetic defects of TJ-altered functionality can be attributed to pro-inflammatory cytokines, which are produced during intestinal inflammation, including interleukin-1β (IL-1β), tumor necrosis factor-α, and interferon-γ, which all have important intestinal TJ barrier-modulating activity ^[59][74]. Another structural alteration that has been described in CD enterocytes of GFD–CD patients is the alteration of vesicular trafficking. CD enterocytes from GFD–CD patients present a constitutive alteration in the intracellular vesicular system at the early recycling compartment level. In CD enterocytes, the number of early vesicles is increased, and EGF/EGFR trafficking is delayed in early endocytic vesicles. Moreover, the decay of EGFR is prolonged, and EEA1 (early endosome Antigen1) and TfR (transferrin receptor) levels are increased ^[23][39]. Recently, several human diseases characterized by inflammation and/or autoimmunity have been attributed to alterations in vesicular trafficking at various levels ^[60][61][75,76]. In particular, hereditary autoimmune-mediated lung disease in humans was found to be due to alterations in vesicular trafficking, revealing a role for intracellular transport in the induction of human autoimmunity ^[62][77]. An analysis of non-HLA loci and/or published eQTL (expression quantitative trait loci) effects associated with CD revealed that 8 out of 127 that had previously been shown to be related to the immune response or other functions were also related to vesicular trafficking ^[23][39]. The results of the analysis of the non-HLA loci and/or published eQTL effects support a role for vesicular trafficking in the pathogenesis of CD. To increase complexity, epigenetic changes such as DNA methylation may also contribute to the mechanisms that lead to the disease or, once the disease has been initiated, establish a chronic disorder. As an example, evaluation of the methylome of the epithelial and immune cell populations of duodenal biopsies in CD patients and healthy individuals showed that these cell populations present different methylation signatures, which may have a broad effect on structural cellular alterations in a cell type-specific manner ^[7][63][23,78]. In CD, the genes linked to inflammatory processes are upregulated, as said before, whereas the genes involved in cell adhesion/integrity of the intestinal barrier are downregulated. All together, expression and methylation studies have highlighted a “gene-expression phenotype” of CD and have shown that the abnormal response to dietary antigens in CD might be related not to abnormalities of gene structure but to the regulation of molecular pathways ^[7][23]. In conclusion, studies on the GFD–CD population have helped to give an important insight into the pathogenesis of CD, but their meaning is still controversial. Some researchers regard the alterations found in GFD–CD patients as an effect of residual amounts of gluten in the diet ^[10][26]. Although small amounts of gluten contaminant cannot be ruled out from food and the CD intestines may be sensitive even to small amounts of gliadin, new insights from compartments far away from the intestines and intestinal organoids point to the presence of constitutive alterations in CD, as discussed in the next paragraphs.

2.3. Intestinal Organoids

An emerging role in CD pathogenesis has been attributed to the intestinal epithelium. In epithelial cells of CD patients, morphological and functional alterations have been described together with the activation of the inflammasome pathway ^[23][64][39,79]. Organoids derived from the small intestine represent a new tool to study the role of the intestinal epithelium in several different diseases ^[65][80]. Intestinal organoids are derived from crypt stem cells cultivated in 3D and embedded in a matrix; they resemble the small intestinal epithelium. Organoids from CD patients have shown the presence of increased staminality, permeability, inflammasome activity, and innate immunity genes with respect to organoids in healthy individuals ^[33][64][48,79]. Instead, extracellular matrix (ECM) genes were decreased in ^[34][49]. Moreover, increased markers of inflammation were found at the protein and mRNA levels in CD organoids. This inflammation was not a residual effect of the tissue of origin but is probably constitutive, as it was persistent even after many days in culture ^[1][17]. In CD biopsies and in intestinal organoids, increased sensitivity to inflammatory stimuli from bacteria ^[64][79], viral ligand loxoribine ^[1][17] and gliadin peptide P31-43 ^[1][17] have been described, indicating that intestinal organoids from CD patients are more sensitive to pro-inflammatory stimuli. Taken all together, these observations indicate that CD intestinal epithelial cells are constitutively different from those in healthy individuals.