TLR3 signaling is downregulated by a MAVS isoform in epithelial cells.

Innate immune responses to dsRNA result in signaling through the TLR3 pathway and/or the RIG-I/MDA-5/MAVS pathway which can activate type I IFN, proinflammatory cytokines and apoptosis. It is not clear whether MAVS could play a role in TLR3-dependent responses to extracellular dsRNA. Using a model of epithelial cells that express a functional TLR3 signaling pathway, we found that TLR3-dependent responses to extracellular dsRNA are negatively regulated by MAVS, precisely "miniMAVS", a recently described 50kDa isoform of MAVS. This regulation of TLR3 by a MAVS isoform constitutes an endogenous regulatory mechanism in epithelial cells that could help prevent a potentially damaging excessive inflammatory response.

Antibody biomarker discovery through in vitro directed evolution of consensus recognition epitopes.

To enable discovery of serum antibodies indicative of disease and simultaneously develop reagents suitable for diagnosis, in vitro directed evolution was applied to identify consensus peptides recognized by patients' serum antibodies. Bacterial cell-displayed peptide libraries were quantitatively screened for binders to serum antibodies from patients with celiac disease (CD), using cell-sorting instrumentation to identify two distinct consensus epitope families specific to CD patients (PEQ and (E)/DxFV(Y)/FQ). Evolution of the (E)/DxFV(Y)/FQ consensus epitope identified a celiac-specific epitope, distinct from the two CD hallmark antigens tissue transglutaminase-2 and deamidated gliadin, exhibiting 71% sensitivity and 99% specificity (n = 231). Expansion of the first-generation PEQ consensus epitope via in vitro evolution yielded octapeptides QPEQAFPE and PFPEQxFP that identified ω- and γ-gliadins, and their deamidated forms, as immunodominant B-cell epitopes in wheat and related cereal proteins. The evolved octapeptides, but not first-generation peptides, discriminated one-way blinded CD and non-CD sera (n = 78) with exceptional accuracy, yielding 100% sensitivity and 98% specificity. Because this method, termed antibody diagnostics via evolution of peptides, does not require prior knowledge of pathobiology, it may be broadly useful for de novo discovery of antibody biomarkers and reagents for their detection.

GM-CSF produced by nonhematopoietic cells is required for early epithelial cell proliferation and repair of injured colonic mucosa.

GM-CSF is a growth factor that promotes the survival and activation of macrophages and granulocytes, as well as dendritic cell differentiation and survival in vitro. The mechanism by which exogenous GM-CSF ameliorates the severity of Crohn's disease in humans and colitis in murine models has mainly been considered to reflect its activity on myeloid cells. We used GM-CSF-deficient (GM-CSF(-/-)) mice to probe the functional role of endogenous host-produced GM-CSF in a colitis model induced after injury to the colon epithelium. Dextran sodium sulfate (DSS), at doses that resulted in little epithelial damage and mucosal ulceration in wild type mice, caused marked colon ulceration and delayed ulcer healing in GM-CSF(-/-) mice. Colon crypt epithelial cell proliferation in vivo was significantly decreased in GM-CSF(-/-) mice at early times after DSS injury. This was paralleled by decreased expression of crypt epithelial cell genes involved in cell cycle, proliferation, and wound healing. Decreased crypt cell proliferation and delayed ulcer healing in GM-CSF(-/-) mice were rescued by exogenous GM-CSF, indicating the lack of a developmental abnormality in the epithelial cell proliferative response in those mice. Nonhematopoietic cells, and not myeloid cells, produced the GM-CSF important for colon epithelial proliferation after DSS-induced injury, as revealed by bone marrow chimera and dendritic cell-depletion experiments, with colon epithelial cells being the cellular source of GM-CSF. Endogenous epithelial cell-produced GM-CSF has a novel nonredundant role in facilitating epithelial cell proliferation and ulcer healing in response to injury of the colon crypt epithelium.

TLR3, TRIF, and caspase 8 determine double-stranded RNA-induced epithelial cell death and survival in vivo.

TLR3 signaling is activated by dsRNA, a virus-associated molecular pattern. Injection of dsRNA into mice induced a rapid, dramatic, and reversible remodeling of the small intestinal mucosa with significant villus shortening. Villus shortening was preceded by increased caspase 3 and 8 activation and apoptosis of intestinal epithelial cells (IECs) located in the mid to upper villus with ensuing luminal fluid accumulation and diarrhea because of an increased secretory state. Mice lacking TLR3 or the adaptor molelcule TRIF mice were completely protected from dsRNA-induced IEC apoptosis, villus shortening, and diarrhea. dsRNA-induced apoptosis was independent of TNF signaling. Notably, NF-κB signaling through IκB kinase ß protected crypt IECs but did not protect villus IECs from dsRNA-induced or TNF-induced apoptosis. dsRNA did not induce early caspase 3 activation with subsequent villus shortening in mice lacking caspase 8 in IECs but instead caused villus destruction with a loss of small intestinal surface epithelium and death. Consistent with direct activation of the TLR3-TRIF-caspase 8 signaling pathway by dsRNA in IECs, dsRNA-induced signaling of apoptosis was independent of non-TLR3 dsRNA signaling pathways, IL-15, TNF, IL-1, IL-6, IFN regulatory factor 3, type I IFN receptor, adaptive immunity, as well as dendritic cells, NK cells, and other hematopoietic cells. We conclude that dsRNA activation of the TLR3-TRIF-caspase 8 signaling pathway in IECs has a significant impact on the structure and function of the small intestinal mucosa and suggest signaling through this pathway has a host protective role during infection with viral pathogens.

Barriers impeding serologic screening for celiac disease in clinically high-prevalence populations.

BACKGROUND: Celiac disease is present in ~1% of the general population in the United States and Europe. Despite the availability of inexpensive serologic screening tests, ~85% of individuals with celiac disease remain undiagnosed and there is an average delay in diagnosis of symptomatic individuals with celiac disease that ranges from ~5.8-11 years. This delay is often attributed to the use of a case-based approach for detection rather than general population screening for celiac disease, and deficiencies at the level of health care professionals. This study aimed to assess if patient-centered barriers have a role in impeding serologic screening for celiac disease in individuals from populations that are clinically at an increased risk for celiac disease. METHODS: 119 adults meeting study inclusion criteria for being at a higher risk for celiac disease were recruited from the general population. Participants completed a survey/questionnaire at the William K. Warren Medical Research Center for Celiac Disease that addressed demographic information, celiac disease related symptoms (gastrointestinal and extraintestinal), family history, co-morbid diseases and conditions associated with celiac disease, and patient-centered barriers to screening for celiac disease. All participants underwent serologic screening for celiac disease using the IgA tissue transglutaminase antibody (IgA tTG) and, if positive, testing for IgA anti-endomysial antibody (IgA EMA) as a confirmatory test. RESULTS: Two barriers to serologic testing were significant across the participant pool. These were participants not knowing they were at risk for celiac disease before learning of the study, and participants not knowing where to get tested for celiac disease. Among participants with incomes less than $25,000/year and those less than the median age, not having a doctor to order the test was a significant barrier, and this strongly correlated with not having health insurance. Symptoms and co-morbid conditions were similar among those whose IgA tTG were negative and those who tested positive. CONCLUSION: There are significant patient-centered barriers that impede serologic screening and contribute to the delayed detection and diagnosis of celiac disease. These barriers may be lessened by greater education of the public and health care professionals about celiac disease symptoms, risk factors, and serologic testing.

Antibody repertoire profiling using bacterial display identifies reactivity signatures of celiac disease.

A general strategy to identify serum antibody specificities associated with a given disease state and peptide reagents for their detection was developed using bacterial display peptide libraries and multiparameter flow cytometry (MPFC). Using sera from patients with celiac disease (CD) (n = 45) or healthy subjects (n = 40), bacterial display libraries were screened for peptides that react specifically with antibodies from CD patients and not with those from healthy patients. The libraries were screened for peptides that simultaneously cross-react with CD patient antibodies present in two separate patient groups labeled with spectrally distinct fluorophores but do not react with unlabeled non-CD antibodies, thus affording a quantitative separation. A panel of six unique peptide sequences yielded 85% sensitivity and 91% specificity (AUC = 0.91) on a set of 60 samples not used for discovery, using leave-one-out cross-validation. Individual peptides were dissimilar with known CD-specific antigens tissue transglutaminase (tTG) and deamidated gliadin, and the classifier accuracy was independent of anti-tTG antibody titer. These results demonstrate that bacterial display/MPFC provides a highly effective tool for the unbiased discovery of disease-associated antibody specificities and peptide reagents for their detection that may have broad utility for diagnostic development.

Maintenance of colonic homeostasis by distinctive apical TLR9 signalling in intestinal epithelial cells.

The mechanisms by which commensal bacteria suppress inflammatory signalling in the gut are still unclear. Here, we present a cellular mechanism whereby the polarity of intestinal epithelial cells (IECs) has a major role in colonic homeostasis. TLR9 activation through apical and basolateral surface domains have distinct transcriptional responses, evident by NF-kappaB activation and cDNA microarray analysis. Whereas basolateral TLR9 signals IkappaBalpha degradation and activation of the NF-kappaB pathway, apical TLR9 stimulation invokes a unique response in which ubiquitinated IkappaB accumulates in the cytoplasm preventing NF-kappaB activation. Furthermore, apical TLR9 stimulation confers intracellular tolerance to subsequent TLR challenges. IECs in TLR9-deficient mice, when compared with wild-type and TLR2-deficient mice, display a lower NF-kappaB activation threshold and these mice are highly susceptible to experimental colitis. Our data provide a case for organ-specific innate immunity in which TLR expression in polarized IECs has uniquely evolved to maintain colonic homeostasis and regulate tolerance and inflammation.

RIG-I/MDA5/MAVS are required to signal a protective IFN response in rotavirus-infected intestinal epithelium.

Rotavirus is a dsRNA virus that infects epithelial cells that line the surface of the small intestine. It causes severe diarrheal illness in children and ∼500,000 deaths per year worldwide. We studied the mechanisms by which intestinal epithelial cells (IECs) sense rotavirus infection and signal IFN-ß production, and investigated the importance of IFN-ß production by IECs for controlling rotavirus production by intestinal epithelium and virus excretion in the feces. In contrast with most RNA viruses, which interact with either retinoic acid-inducible gene I (RIG-I) or melanoma differentiation-associated gene 5 (MDA5) inside cells, rotavirus was sensed by both RIG-I and MDA5, alone and in combination. Rotavirus did not signal IFN-ß through either of the dsRNA sensors TLR3 or dsRNA-activated protein kinase (PKR). Silencing RIG-I or MDA5, or their common adaptor protein mitochondrial antiviral signaling protein (MAVS), significantly decreased IFN-ß production and increased rotavirus titers in infected IECs. Overexpression of laboratory of genetics and physiology 2, a RIG-I-like receptor that interacts with viral RNA but lacks the caspase activation and recruitment domains required for signaling through MAVS, significantly decreased IFN-ß production and increased rotavirus titers in infected IECs. Rotavirus-infected mice lacking MAVS, but not those lacking TLR3, TRIF, or PKR, produced significantly less IFN-ß and increased amounts of virus in the intestinal epithelium, and shed increased quantities of virus in the feces. We conclude that RIG-I or MDA5 signaling through MAVS is required for the activation of IFN-ß production by rotavirus-infected IECs and has a functionally important role in determining the magnitude of rotavirus replication in the intestinal epithelium.

The two faces of IKK and NF-kappaB inhibition: prevention of systemic inflammation but increased local injury following intestinal ischemia-reperfusion.

We studied the role of NF-kappaB in acute inflammation caused by gut ischemia-reperfusion through selective ablation of IkappaB kinase (IKK)-beta, the catalytic subunit of IKK that is essential for NF-kappaB activation. Ablation of IKK-beta in enterocytes prevented the systemic inflammatory response, which culminates in multiple organ dysfunction syndrome (MODS) that is normally triggered by gut ischemia-reperfusion. IKK-beta removal from enterocytes, however, also resulted in severe apoptotic damage to the reperfused intestinal mucosa. These results show the dual function of the NF-kappaB system, which is responsible for both tissue protection and systemic inflammation, and underscore the caution that should be exerted in using NF-kappaB and IKK inhibitors.

Opposing functions of IKKbeta during acute and chronic intestinal inflammation.

NF-kappaB is a key transcriptional regulator of inflammatory responses, but also controls expression of prosurvival genes, whose products protect tissues from damage and may thus act indirectly in an antiinflammatory fashion. The variable importance of these two distinct NF-kappaB-controlled responses impacts the potential utility of NF-kappaB inhibition as a treatment strategy for intractable inflammatory conditions, such as inflammatory bowel disease. Here, we show in murine models that inhibition of IKKbeta-dependent NF-kappaB activation exacerbates acute inflammation, but attenuates chronic inflammatory disease in the intestinal tract. Acute ulcerating inflammation is aggravated because of diminished NF-kappaB-mediated protection against epithelial cell apoptosis and delayed mucosal regeneration secondary to reduced NF-kappaB-dependent recruitment of inflammatory cells that secrete cytoprotective factors. In contrast, in IL-10-deficient mice, which serve as a model of chronic T cell-dependent colitis, ablation of IKKbeta in the intestinal epithelium has no impact, yet IKKbeta deficiency in myeloid cells attenuates inflammation and prolongs survival. These results highlight the striking context and tissue dependence of the proinflammatory and antiapoptotic functions of NF-kappaB. Our findings caution against the therapeutic use of IKKbeta/NF-kappaB inhibitors in acute inflammatory settings dominated by cell loss and ulceration.

Celiac disease: pathogenesis of a model immunogenetic disease.

Celiac disease is characterized by small-intestinal mucosal injury and nutrient malabsorption in genetically susceptible individuals in response to the dietary ingestion of wheat gluten and similar proteins in barley and rye. Disease pathogenesis involves interactions among environmental, genetic, and immunological factors. Although celiac disease is predicted by screening studies to affect approximately 1% of the population of the United States and is seen both in children and in adults, 10%-15% or fewer of these individuals have been diagnosed and treated. This article focuses on the role of adaptive and innate immune mechanisms in the pathogenesis of celiac disease and how current concepts of immunopathogenesis might provide alternative approaches for treating celiac disease.

Stool antigen immunodetection for diagnosis of Giardia duodenalis infection in human subjects with HIV and cancer.

Human infection with the protozoan parasite Giardia duodenalis is one the most common parasitic diseases worldwide. Higher incidence rates of giardiasis have been reported from human subjects with multiple debilitating chronic conditions, including hypogammaglobulinemia and common variable immunodeficiency (CVID). In the current study, stool specimens were collected from 199 individuals diagnosed with HIV or cancer and immunocompetent subjects. The sensitivity of microscopy-based detection on fresh stool preparations, trichrome staining and stool antigen immunodetection for the diagnosis of G. duodenalis were 36%, 45.5% and 100%, respectively when compared with a highly sensitive stool-based PCR method as the gold standard. Further multilocus molecular analyses using glutamate dehydrogenase (gdh) and triose phosphate isomerase (tpi) loci demonstrated that the AI genotype of G. duodenalis was the most prevalent, followed by the AII genotype and mixed (AI+B) infections. We concluded that stool antigen immunodetection-based immunoassays and stool-based PCR amplification had comparable sensitivity and specificity for the diagnosis of G. duodenalis infections in these populations. Stool antigen detection-based diagnostic modalities are rapid and accurate and may offer alternatives to conventional microscopy and PCR-based diagnostic methods for the diagnosis of G. duodenalis in human subjects living with HIV or cancer.

Recent understanding of IBD pathogenesis: implications for future therapies.

The inflammatory bowel diseases (IBD) are comprised of two major phenotypes, Crohn's disease (CD) and ulcerative colitis (UC). Research over the last couple of years has led to great advances in understanding the inflammatory bowel diseases and their underlying pathophysiologic mechanisms. From the current understanding, it is likely that chronic inflammation in IBD is due to aggressive cellular immune responses to a subset of luminal bacteria. Susceptibility to disease is thereby determined by genes encoding immune responses which are triggered by environmental stimuli. Based on extensive research over the last decade, there are several new and novel pathways and specific targets on which to focus new therapeutics. The following review summarizes the current view on the four basic tenets of the pathophysiological basis of IBD and its implications for therapies of IBD: genetics, immune dysregulation, barrier dysfunction and the role of the microbial flora.

Microbial-epithelial cell crosstalk during inflammation: the host response.

The intestinal epithelium forms a single cell barrier that separates the host's internal milieu from luminal contents. This article examines the role of the intestinal epithelium as a critical component of a communications network that is essential for transmitting signals generated in response to infection with microbial pathogens to cells of the innate and acquired immune systems in the underlying intestinal mucosa. It further highlights the importance of intestinal epithelium in mediating host antimicrobial defense through the production of antimicrobial peptides of the defensin and cathelicidin families.

Adaptive immune response in symptomatic and asymptomatic enteric protozoal infection: evidence for a determining role of parasite genetic heterogeneity in host immunity to human giardiasis.

The genetic basis of the ultimate clinical outcomes of human giardiasis has been the subject of numerous investigations. We previously demonstrated roles for both host and parasite factors in determining the outcome of enteric infection in a murine model of Giardia duodenalis infection. In the current study, fecal and serum specimens from healthy controls and human subjects infected with the intestinal parasite G. duodenalis were assessed. Using a semi-nested PCR method, clinical isolates were genetically characterized based on the gdh and tpi loci, and the phylogenetic trees were constructed. Using a sandwich ELISA method, the serum levels of representative TH1 and TH2 cytokines were measured in infected human subjects and healthy controls. Here we showed that symptomatic human giardiasis was characterized by significantly elevated serum levels of the TH1 cytokine IFN-γ compared to healthy controls, whereas asymptomatic human subjects and healthy controls had comparable levels of serum IFN-γ. Further analyses showed that human subjects infected with G. duodenalis genotype AI had significantly elevated levels of serum IFN-γ and IL-10, but not IL-5, whereas human subjects infected with AII had similar levels of those cytokines compared to healthy controls. These data demonstrate roles for both host and parasite factors in the determination of the outcome of enteric infections and may further broaden our understanding of host-parasite interaction during enteric protozoal infections.

Lack of the programmed death-1 receptor renders host susceptible to enteric microbial infection through impairing the production of the mucosal natural killer cell effector molecules.

The programmed death-1 receptor is expressed on a wide range of immune effector cells, including T cells, natural killer T cells, dendritic cells, macrophages, and natural killer cells. In malignancies and chronic viral infections, increased expression of programmed death-1 by T cells is generally associated with a poor prognosis. However, its role in early host microbial defense at the intestinal mucosa is not well understood. We report that programmed death-1 expression is increased on conventional natural killer cells but not on CD4(+), CD8(+) or natural killer T cells, or CD11b(+) or CD11c(+) macrophages or dendritic cells after infection with the mouse pathogen Citrobacter rodentium. Mice genetically deficient in programmed death-1 or treated with anti-programmed death-1 antibody were more susceptible to acute enteric and systemic infection with Citrobacter rodentium. Wild-type but not programmed death-1-deficient mice infected with Citrobacter rodentium showed significantly increased expression of the conventional mucosal NK cell effector molecules granzyme B and perforin. In contrast, natural killer cells from programmed death-1-deficient mice had impaired expression of those mediators. Consistent with programmed death-1 being important for intracellular expression of natural killer cell effector molecules, mice depleted of natural killer cells and perforin-deficient mice manifested increased susceptibility to acute enteric infection with Citrobacter rodentium. Our findings suggest that increased programmed death-1 signaling pathway expression by conventional natural killer cells promotes host protection at the intestinal mucosa during acute infection with a bacterial gut pathogen by enhancing the expression and production of important effectors of natural killer cell function.

The intestinal epithelium is an integral component of a communications network.

The epithelial lining of the intestine forms a barrier that separates the intestinal lumen from the host's internal milieu and is critical for fluid and electrolyte secretion and nutrient absorption. In the early 1990s, my laboratory discovered that intestinal epithelial cells could alter their phenotype and produce proinflammatory chemokines and cytokines when stimulated by pathogenic enteric luminal microbes or proinflammatory agonists produced by cells in the underlying mucosa. It is now well accepted that intestinal epithelial cells can be induced to express and secrete specific arrays of cytokines, chemokines, and antimicrobial defense molecules. The coordinated release of molecules by intestinal epithelial cells is crucial for activating intestinal mucosal inflammatory responses as well as mucosal innate and adaptive immune responses. More recent studies have focused on the intestinal epithelial signaling pathways that culminate in immune activation as well as the role of these pathways in host defense, mucosal injury, mucosal wound healing, and tumorigenesis. The emerging picture indicates that intestinal epithelial cells represent an integral component of a highly regulated communications network that can transmit essential signals to cells in the underlying intestinal mucosa, and that intestinal epithelial cells, in turn, serve as targets of mucosal mediators. These signals are essential for maintaining intestinal mucosal defense and homeostasis.

Introduction: celiac disease.

This is the introduction for the Special Issue on Celiac Disease.

The immunopathogenesis of celiac disease reveals possible therapies beyond the gluten-free diet.

Celiac disease is a T cell-mediated autoimmune inflammatory disease of the small intestine that is activated by gluten. The diagnosis of celiac disease is challenging as patients display a wide range of symptoms and some are asymptomatic. A lifelong gluten-free diet is the only currently approved treatment of celiac disease. Although the diet is safe and effective, the compliance rates and patient acceptance vary. Furthermore, many patients treated with a gluten-free diet continue to be mildly to severely symptomatic with persistent histological abnormalities, and a small number of patients develop refractory celiac disease. New therapeutic adjuncts and potential alternatives to the gluten-free diet could improve the treatment options for these patients. Advances in understanding the immunopathogenesis of celiac disease have suggested several types of therapeutic strategies that may augment or supplant the gluten-free diet. Some of these strategies attempt to decrease the immunogenicity of gluten-containing grains by manipulating the grain itself or by using oral enzymes to break down immunogenic peptides that normally remain intact during digestion. Other strategies focus on preventing the absorption of these peptides, preventing tissue transglutaminase from rendering gluten peptides more immunogenic, or inhibiting their binding to celiac disease-specific antigen-presenting molecules. Strategies that limit T cell migration to the small intestine or that reestablish mucosal homeostasis and tolerance to gluten antigens are also being explored. Additionally, it is vital to develop new therapeutic options for refractory celiac disease patients. This review highlights therapeutic strategies that may ultimately improve the health and well-being of individuals with celiac disease.

Can consumers trust web-based information about celiac disease? Accuracy, comprehensiveness, transparency, and readability of information on the internet.

BACKGROUND: Celiac disease is an autoimmune disease that affects approximately 1% of the US population. Disease is characterized by damage to the small intestinal lining and malabsorption of nutrients. Celiac disease is activated in genetically susceptible individuals by dietary exposure to gluten in wheat and gluten-like proteins in rye and barley. Symptoms are diverse and include gastrointestinal and extraintestinal manifestations. Treatment requires strict adherence to a gluten-free diet. The Internet is a major source of health information about celiac disease. Nonetheless, information about celiac disease that is available on various websites often is questioned by patients and other health care professionals regarding its reliability and content. OBJECTIVES: To determine the accuracy, comprehensiveness, transparency, and readability of information on 100 of the most widely accessed websites that provide information on celiac disease. METHODS: Using the search term celiac disease, we analyzed 100 of the top English-language websites published by academic, commercial, nonprofit, and other professional (nonacademic) sources for accuracy, comprehensiveness, transparency, and reading grade level. Each site was assessed independently by 3 reviewers. Website accuracy and comprehensiveness were probed independently using a set of objective core information about celiac disease. We used 19 general criteria to assess website transparency. Website readability was determined by the Flesch-Kincaid reading grade level. Results for each parameter were analyzed independently. In addition, we weighted and combined parameters to generate an overall score, termed website quality. RESULTS: We included 98 websites in the final analysis. Of these, 47 (48%) provided specific information about celiac disease that was less than 95% accurate (ie, the predetermined cut-off considered a minimum acceptable level of accuracy). Independent of whether the information posted was accurate, 51 of 98 (52%) websites contained less than 50% of the core celiac disease information that was considered important for inclusion on websites that provide general information about celiac disease. Academic websites were significantly less transparent (P = .005) than commercial websites in attributing authorship, timeliness of information, sources of information, and other important disclosures. The type of website publisher did not predict website accuracy, comprehensiveness, or overall website quality. Only 4 of 98 (4%) websites achieved an overall quality score of 80 or above, which a priori was set as the minimum score for a website to be judged trustworthy and reliable. CONCLUSIONS: The information on many websites addressing celiac disease was not sufficiently accurate, comprehensive, and transparent, or presented at an appropriate reading grade level, to be considered sufficiently trustworthy and reliable for patients, health care providers, celiac disease support groups, and the general public. This has the potential to adversely affect decision making about important aspects of celiac disease, including its appropriate and proper diagnosis, treatment, and management.