A sensitive flow cytometric method for multi-parametric analysis of microRNA, messenger RNA and protein in single cells.

Analysis of RNA expression in mixed cell populations often requires laborious and costly cell sorting. Here we describe a flow cytometric assay that combines antibody staining and in situ hybridization for multi-parametric analysis of single cells. This method, referred to as the PrimeFlow™ RNA Assay, enables simultaneous detection of protein markers and RNA targets in mixed cell populations. Both coding and non-coding RNA sequences can be measured with a limit of detection of approximately 10 copies of mRNA and 20 copies of microRNA per cell. In this study, we used mouse bone marrow-derived macrophages to demonstrate that our method allows for analysis of the activation and polarization status of cells using expression patterns of protein and RNA. We then performed analysis of four cell subsets of mouse resident peritoneal cells and showed that the two macrophage populations present in this compartment are relatively heterogeneous in terms of expression of two M2 markers: Arg1, Retnla, and a B-cell attractant chemokine Cxcl13. In addition, we profiled the expression of a panel of microRNA in the four peritoneal cell subsets, showing that the assay can be readily adapted to parallel, high-throughput screening of multiple cell populations. This new method allows for single cell analysis of multiple RNA targets without the need for cell sorting, enables direct correlation between RNA and protein expression, and promises to accelerate biomarker and drug discovery.

αßT cell receptors expressed by CD4(-)CD8αß(-) intraepithelial T cells drive their fate into a unique lineage with unusual MHC reactivities.

Coreceptor CD4 and CD8αß double-negative (DN) TCRαß(+) intraepithelial T cells, although numerous, have been greatly overlooked and their contribution to the immune response is not known. Here we used T cell receptor (TCR) sequencing of single cells combined with retrogenic expression of TCRs to study the fate and the major histocompatibility complex (MHC) restriction of DN TCRαß(+) intraepithelial T cells. The data show that commitment of thymic precursors to the DN TCRαß(+) lineage is imprinted by their TCR specificity. Moreover, the TCRs they express display a diverse and unusual pattern of MHC restriction that is nonoverlapping with that of CD4(+) or CD8αß(+) T cells, indicating that they sense antigens that are not recognized by the conventional T cell subsets. The new insights indicate that DN TCRαß(+) T cells form a third lineage of TCRαß T lymphocytes expressing a variable TCR repertoire, which serve nonredundant immune functions.

Intestinal microbes affect phenotypes and functions of invariant natural killer T cells in mice.

BACKGROUND & AIMS: Invariant natural killer T (iNKT) cells undergo canonical, Vα14-Jα18 rearrangement of the T-cell receptor (TCR) in mice; this form of the TCR recognizes glycolipids presented by CD1d. iNKT cells mediate many different immune reactions. Their constitutive activated and memory phenotype and rapid initiation of effector functions after stimulation indicate previous antigen-specific stimulation. However, little is known about this process. We investigated whether symbiotic microbes can determine the activated phenotype and function of iNKT cells. METHODS: We analyzed the numbers, phenotypes, and functions of iNKT cells in germ-free mice, germ-free mice reconstituted with specified bacteria, and mice housed in specific pathogen-free environments. RESULTS: Specific pathogen-free mice, obtained from different vendors, have different intestinal microbiota. iNKT cells isolated from these mice differed in TCR Vß7 frequency and cytokine response to antigen, which depended on the environment. iNKT cells isolated from germ-free mice had a less mature phenotype and were hyporesponsive to activation with the antigen α-galactosylceramide. Intragastric exposure of germ-free mice to Sphingomonas bacteria, which carry iNKT cell antigens, fully established phenotypic maturity of iNKT cells. In contrast, reconstitution with Escherichia coli, which lack specific antigens for iNKT cells, did not affect the phenotype of iNKT cells. The effects of intestinal microbes on iNKT cell responsiveness did not require Toll-like receptor signals, which can activate iNKT cells independently of TCR stimulation. CONCLUSIONS: Intestinal microbes can affect iNKT cell phenotypes and functions in mice.

Constitutive intestinal NF-κB does not trigger destructive inflammation unless accompanied by MAPK activation.

Nuclear factor (NF)-κB, activated by IκB kinase (IKK), is a key regulator of inflammation, innate immunity, and tissue integrity. NF-κB and one of its main activators and transcriptional targets, tumor necrosis factor (TNF), are up-regulated in many inflammatory diseases that are accompanied by tissue destruction. The etiology of many inflammatory diseases is poorly understood, but often depends on genetic factors and environmental triggers that affect NF-κB and related pathways. It is unknown, however, whether persistent NF-κB activation is sufficient for driving symptomatic chronic inflammation and tissue damage. To address this question, we generated IKKß(EE)(IEC) mice, which express a constitutively active form of IKKß in intestinal epithelial cell (IECs). IKKß(EE)(IEC) mice exhibit NF-κB activation in IECs and express copious amounts of inflammatory chemokines, but only small amounts of TNF. Although IKKß(EE)(IEC) mice exhibit inflammatory cell infiltration in the lamina propria (LP) of their small intestine, they do not manifest tissue damage. Yet, upon challenge with relatively mild immune and microbial stimuli, IKKß(EE)(IEC) mice succumb to destructive acute inflammation accompanied by enterocyte apoptosis, intestinal barrier disruption, and bacterial translocation. Inflammation is driven by massive TNF production, which requires additional activation of p38 and extracellular-signal-regulated kinase mitogen-activated protein kinases (MAPKs).

Dominance of an alternative CLIP sequence in the celiac disease associated HLA-DQ2 molecule.

During assembly, HLA class II molecules associate with the invariant chain. As the result, the peptide-binding groove is occupied by an invariant chain peptide termed CLIP (class-II-associated invariant chain peptide; sequence MRMATPLLM). By mass spectrometry, we have now characterized peptides that are naturally present in HLA-DQ2. This analysis revealed that 22 variants of Ii-derived peptides are associated with HLA-DQ2. Strikingly, the large majority of those do not contain the conventional CLIP sequence MRMATPLLM, but instead a peptide that partially overlaps with CLIP, sequence TPLLMQALPM. Peptide binding studies indicate that this alternative CLIP peptide has superior HLA-DQ2 binding properties compared to the conventional CLIP and that the minimal nine-amino-acid binding core consists of the sequence PLLMQALPM, findings that could be corroborated by molecular simulation. The alternative CLIP peptide was also found to be present in HLA-DQ2 molecules isolated from human thymus. Moreover, the alternative CLIP peptide was also found in association with HLA-DQ8. Together, these results indicate that HLA-DQ2 and HLA-DQ8 associate with an alternative CLIP sequence, a property that may relate to the strong association between HLA-DQ molecules and human autoimmune diseases.

Large-scale characterization of natural ligands explains the unique gluten-binding properties of HLA-DQ2.

Celiac disease is an enteropathy caused by intolerance to dietary gluten. The disorder is strongly associated with DQA1*0501/DQB1*0201 (HLA-DQ2) as approximately 95% of celiac patients express this molecule. HLA-DQ2 has unique Ag-binding properties that allow it to present a diverse set of gluten peptides to gluten-reactive CD4+ T cells so instigating an inflammatory reaction. Previous work has indicated that the presence of negatively charged amino acids within gluten peptides is required for specific binding. This, however, only partly explains the scale of the interaction. We have now characterized 432 natural ligands of HLA-DQ2 representing length variants of 155 distinct sequences. The sequences were aligned and the binding cores were inferred. Analysis of the amino acid distribution of these cores demonstrated that negatively charged residues in HLA-DQ2-bound peptides are favored at virtually all positions. This contrasts with a more restricted presence of such amino acids in T cell epitopes from gluten. Yet, HLA-DQ2 was also found to display a strong preference for proline at several anchor and nonanchor positions that largely match the position of proline in gluten T cell epitopes. Consequently, the bias for proline at p6 and p8 facilitates the enzymatic conversion of glutamine into glutamic acid in gluten peptides at p4 and p6, two important anchor sites. These observations provide new insights in the unique ability of HLA-DQ2 to bind a large repertoire of glutamine- and proline-rich gluten peptides. This knowledge may be an important asset in the development of future treatment strategies.

Enzymatic gluten detoxification: the proof of the pudding is in the eating!

Celiac disease is caused by an immune response to the dietary protein gluten. The only available treatment is the strict exclusion of gluten from the diet; however, this is marred by the virtual omnipresence of this protein. The enzymatic degradation of gluten might become an alternative to the gluten-free diet, and recent work indicates that such approaches are getting close to being tested in clinical trials.

Genetic and functional analysis of pyroglutamyl-peptidase I in coeliac disease.

Coeliac disease (CD) is an enteropathy caused by an immune reaction towards wheat gluten and similar proteins from barley and rye. It was shown that some gluten peptides spontaneously form N-terminal L-pyroglutamate. This modification could potentially make gluten more resistant to proteolytic degradation within the intestine. Pyroglutamyl-peptidase I (PGPEPI) is an enzyme that hydrolytically removes the L-pyroglutamyl residues that render the modified proteins and peptides more sensitive to degradation by other proteases. Interestingly, we found that the PGPEP1 gene is located in a CD susceptibility locus. As an impaired enzyme function caused by genetic alterations might increase the amount of immunogenic gluten peptides, we conducted a comprehensive functional genomics analysis of PGPEP1, including DNA sequencing, genetic association testing, and quantifying RNA expression. We also determined the enzymatic activity of PGPEPI in duodenal biopsies. Our results uniformly indicate that PGPEP1 is not involved in the aetiology and pathology of CD.

Highly efficient gluten degradation with a newly identified prolyl endoprotease: implications for celiac disease.

Celiac disease is a T cell-driven intolerance to wheat gluten. The gluten-derived T cell epitopes are proline-rich and thereby highly resistant to proteolytic degradation within the gastrointestinal tract. Oral supplementation with prolyl oligopeptidases has therefore been proposed as a potential therapeutic approach. The enzymes studied, however, have limitations as they are irreversibly inactivated by pepsin and acidic pH, both present in the stomach. As a consequence, these enzymes will fail to degrade gluten before it reaches the small intestine, the site where gluten induces inflammatory T cell responses that lead to celiac disease. We have now determined the usefulness of a newly identified prolyl endoprotease from Aspergillus niger for this purpose. Gluten and its peptic/tryptic digest were treated with prolyl endoprotease, and the destruction of the T cell epitopes was tested using mass spectrometry, T cell proliferation assays, ELISA, reverse-phase HPLC, SDS-PAGE, and Western blotting. We observed that the A. niger prolyl endoprotease works optimally at 4-5 pH, remains stable at 2 pH, and is completely resistant to digestion with pepsin. Moreover, the A. niger-derived enzyme efficiently degraded all tested T cell stimulatory peptides as well as intact gluten molecules. On average, the endoprotease from A. niger degraded gluten peptides 60 times faster than a prolyl oligopeptidase. Together these results indicate that the enzyme from A. niger efficiently degrades gluten proteins. Future studies are required to determine if the prolyl endoprotease can be used as an oral supplement to reduce gluten intake in patients.

Celiac disease--sandwiched between innate and adaptive immunity.

Celiac disease (CD) patients are intolerant to gluten, proteins in wheat, and related cereals. Virtually all patients are human leukocyte antigen (HLA)-DQ2 or HLA-DQ8 positive and several studies have demonstrated that CD4 T cells specific for (modified) gluten peptides bound to these HLA-DQ molecules are found in patients but not in control subjects. These T cell responses are therefore thought to be responsible for disease development. Many immunogenic gluten peptides which may relate to the disease-inducing properties of gluten have now been identified. In addition, gluten can stimulate IL-15 production that ultimately leads to NKG2D-mediated epithelial cell killing. However, CD develops in only a minority of HLA-DQ2 and HLA-DQ8 individuals. This may be attributed to the default setting of the intestinal immune system: induction and maintenance of tolerance to dietary components and commensal flora. Although at present it is unknown why tolerance in CD is not established or broken, both environmental and genetic factors have been implicated. There is strong evidence for the existence of genes or gene variants on chromosomes 5, 6, and 19 that predispose to CD. In addition, type I interferons have been implicated in development of several autoimmune disorders, including CD. Thus, viral infection and/or tissue damage in the intestine may cause inflammation and induce protective Th1-mediated immunity leading to loss of tolerance for gluten. Once tolerance is broken, a broad gluten-reactive T cell repertoire may develop through determinant spreading. This may be a critical step toward full-blown disease.

No genetic association of the human prolyl endopeptidase gene in the Dutch celiac disease population.

Celiac disease (CD) is a complex genetic disorder of the small intestine. The DQ2/DQ8 human leucocyte antigen (HLA) genes explain approximately 40% of the genetic component of the disease, but the remaining non-HLA genes have not yet been identified. The key environmental factor known to be involved in the disease is gluten, a major protein present in wheat, barley, and rye. Integrating microarray data and linkage data from chromosome 6q21-22 revealed the prolyl endopeptidase (PREP) gene as a potential CD candidate in the Dutch population. Interestingly, this gene encodes for the only enzyme that is able to cleave the proline-rich gluten peptides. To investigate the role of the human PREP gene as a primary genetic factor in CD, we conducted gene expression, sequence analysis, and genetic association studies of the PREP gene and determined PREP enzyme activity in biopsies from CD patients and controls. Sequence analysis of the coding region of the PREP gene revealed two novel polymorphisms. Genetic association studies using two novel polymorphisms and three known PREP variants excluded a genetic association between PREP and CD. Determination of PREP activity revealed weak but significant differences between treated and untreated CD biopsies (P < 0.05). Our results from the association study indicate that PREP is not a causative gene for CD in the Dutch population. These are further supported by the activity determinations in which we observed no differences in PREP activity between CD patients and controls.

T-cell recognition of HLA-DQ2-bound gluten peptides can be influenced by an N-terminal proline at p-1.

Recent research has implicated a large number of gluten-derived peptides in the pathogenesis of celiac disease, a preponderantly HLA-DQ2-associated disorder. Current evidence indicates that the core of some of those peptides is ten amino acids long, while HLA class II normally accommodates nine amino acids in the binding groove. We have now investigated this in detail, using gluten-specific T-cell clones, HLA-DQ2-specific peptide-binding assays and molecular modelling. T-cell recognition of both a gamma-gliadin peptide and a low-molecular-weight glutenin peptide was found to be strictly dependent on a ten-amino acids-long peptide. Subsequent peptide-binding studies indicated that the glutenin peptide bound in a conventional p1/p9 register, with an additional proline at p-1. Testing of substitution analogues demonstrated that the nature of the amino acid at p-1 strongly influenced T-cell recognition of the peptide. Moreover, molecular modelling confirmed that the glutenin peptide binds in a p1/p9 register, and that the proline at p-1 points upward towards the T-cell receptor. Database searches indicate that a large number of potential T-cell stimulatory gluten peptides with an additional proline at relative position p-1 exist, suggesting that the recognition of other gluten peptides may depend on this proline as well. This knowledge may be of importance for the identification of additional T-cell stimulatory gluten peptides and the design of a peptide-based, tolerance-inducing therapy.

Subcellular sites for bacterial protein export.

Most bacterial proteins destined to leave the cytoplasm are exported to extracellular compartments or imported into the cytoplasmic membrane via the highly conserved SecA-YEG pathway. In the present studies, the subcellular distributions of core components of this pathway, SecA and SecY, and of the secretory protein pre-AmyQ, were analysed using green fluorescent protein fusions, immunostaining and/or immunogold labelling techniques. It is shown that SecA, SecY and (pre-)AmyQ are located at specific sites near and/or in the cytoplasmic membrane of Bacillus subtilis. The localization patterns of these proteins suggest that the Sec machinery is organized in spiral-like structures along the cell, with most of the translocases organized in specific clusters along these structures. However, this localization appears to be independent of the helicoidal structures formed by the actin-like cytoskeletal proteins, MreB or Mbl. Interestingly, the specific localization of SecA is dynamic, and depends on active translation. Moreover, reducing the phosphatidylglycerol phospholipids content in the bacterial membrane results in delocalization of SecA, suggesting the involvement of membrane phospholipids in the localization process. These data show for the first time that, in contrast to the recently reported uni-ExPortal site in the coccoïd Streptococcus pyogenes, multiple sites dedicated to protein export are present in the cytoplasmic membrane of rod-shaped B. subtilis.

The HLA-DQ2 gene dose effect in celiac disease is directly related to the magnitude and breadth of gluten-specific T cell responses.

In patients with celiac disease, inflammatory T cell responses to HLA-DQ2-bound gluten peptides are thought to cause disease. Two types of HLA-DQ2 molecules exist, termed HLA-DQ2.5 and HLA-DQ2.2. Whereas HLA-DQ2.5 predisposes to celiac disease, HLA-DQ2.2 does not. We now provide evidence that the disease-associated HLA-DQ2.5 molecule presents a large repertoire of gluten peptides, whereas the non-disease-associated HLA-DQ2.2 molecule can present only a subset of these. Moreover, gluten presentation by HLA-DQ2 homozygous antigen-presenting cells was superior to presentation by HLA-DQ2/non-DQ2 heterozygous antigen-presenting cells in terms of T cell proliferation and cytokine secretion. Gluten presentation by HLA-DQ2.5/2.2 heterozygous antigen-presenting cells induced intermediate T cell stimulation. These results correlated with peptide binding to the antigen-presenting cells. Finally, we demonstrate that HLA-DQ trans dimers formed in HLA-DQ2.5/2.2 heterozygous individuals have properties identical with HLA-DQ2.5 dimers. Our findings explain the strongly increased risk of disease development for HLA-DQ2.5 homozygous and HLA-DQ2.2/2.5 heterozygous individuals, and they are indicative of a quantitative model for disease development, where HLA-DQ expression and the available number of T cell-stimulatory gluten peptides are critical limiting factors. This model may have important implications for disease prevention.

Characterization of cereal toxicity for celiac disease patients based on protein homology in grains.

BACKGROUND AND AIMS: Celiac disease is caused by T-cell responses to wheat gluten-derived peptides. The presence of such peptides in other widely consumed grains, however, has hardly been studied. METHODS: We have performed homology searches to identify regions with sequence similarity to T-cell stimulatory gluten peptides in the available gluten sequences: the hordeins of barley, secalins of rye, and avenins of oats. The identified peptides were tested for T-cell stimulatory properties. RESULTS: With 1 exception, no identical matches with T-cell stimulatory gluten peptides were found in the other grains. However, less stringent searches identified 11 homologous sequences in hordeins, secalins, and avenins located in regions similar to those in the original gluten proteins. Seven of these 11 peptides were recognized by gluten-specific T-cell lines and/or clones from patients with celiac disease. Comparison of T-cell stimulatory sequences with homologous but non-T-cell stimulatory sequences indicated key amino acids that on substitution either completely or partially abrogated the T-cell stimulatory activity of the gluten peptides. Finally, we show that single nucleotide substitutions in gluten genes will suffice to induce these effects. CONCLUSIONS: These results show that the disease-inducing properties of barley and rye can in part be explained by T-cell cross-reactivity against gluten-, secalin-, and hordein-derived peptides. Moreover, the results provide a first step toward a rational strategy for gluten detoxification via targeted mutagenesis at the genetic level.