Bi-directional signaling between the intestinal epithelium and type-3 innate lymphoid cells regulates secretory dynamics and interleukin-22.

Type-3 innate lymphoid cells (ILC3) respond to localized environmental cues to regulate homeostasis and orchestrate immunity in the intestine. The intestinal epithelium is an important upstream regulator and downstream target of ILC3 signaling, however, the complexity of mucosal tissues can hinder efforts to define specific interactions between these two compartments. Here, we employ a reductionist co-culture system of murine epithelial small intestinal organoids (SIO) with ILC3 to uncover bi-directional signaling mechanisms that underlie intestinal homeostasis. We report that ILC3 induce global transcriptional changes in intestinal epithelial cells, driving the enrichment of secretory goblet cell signatures. We find that SIO enriched for goblet cells promote NKp46+ ILC3 and interleukin (IL)-22 expression, which can feedback to induce IL-22-mediated epithelial transcriptional signatures. However, we show that epithelial regulation of ILC3 in this system is contact-dependent and demonstrate a role for epithelial Delta-Like-Canonical-Notch-Ligand (Dll) in driving IL-22 production by ILC3, via subset-specific Notch1-mediated activation of T-bet+ ILC3. Finally, by interfering with Notch ligand-receptor dynamics, ILC3 appear to upregulate epithelial Atoh1 to skew secretory lineage determination in SIO-ILC3 co-cultures. This research outlines two complimentary bi-directional signaling modules between the intestinal epithelium and ILC3, which may be relevant in intestinal homeostasis and disease.

Automating Assignment, Quantitation, and Biological Annotation of Redox Proteomics Datasets with ProteoSushi.

Redox proteomics plays an increasingly important role characterizing the cellular redox state and redox signaling networks. As these datasets grow larger and identify more redox regulated sites in proteins, they provide a systems-wide characterization of redox regulation across cellular organelles and regulatory networks. However, these large proteomic datasets require substantial data processing and analysis in order to fully interpret and comprehend the biological impact of oxidative posttranslational modifications. We therefore developed ProteoSushi, a software tool to biologically annotate and quantify redox proteomics and other modification-specific proteomics datasets. ProteoSushi can be applied to differentially alkylated samples to assay overall cysteine oxidation, chemically labeled samples such as those used to profile the cysteine sulfenome, or any oxidative posttranslational modification on any residue.Here we demonstrate how to use ProteoSushi to analyze a large, public cysteine redox proteomics dataset. ProteoSushi assigns each modified peptide to shared proteins and genes, sums or averages signal intensities for each modified site of interest, and annotates each modified site with the most up-to-date biological information available from UniProt. These biological annotations include known functional roles or modifications of the site, the protein domain(s) that the site resides in, the protein's subcellular location and function, and more.

Enterotoxigenic Escherichia coli Degrades the Host MUC2 Mucin Barrier To Facilitate Critical Pathogen-Enterocyte Interactions in Human Small Intestine.

Enterotoxigenic Escherichia coli (ETEC) isolates are genetically diverse pathological variants of E. coli defined by the production of heat-labile (LT) and/or heat-stable (ST) toxins. ETEC strains are estimated to cause hundreds of millions of cases of diarrheal illness annually. However, it is not clear that all strains are equally equipped to cause disease, and asymptomatic colonization with ETEC is common in low- to middle-income regions lacking basic sanitation and clean water where ETEC are ubiquitous. Recent molecular epidemiology studies have revealed a significant association between strains that produce EatA, a secreted autotransporter protein, and the development of symptomatic infection. Here, we demonstrate that LT stimulates production of MUC2 mucin by goblet cells in human small intestine, enhancing the protective barrier between pathogens and enterocytes. In contrast, using explants of human small intestine as well as small intestinal enteroids, we show that EatA counters this host defense by engaging and degrading the MUC2 mucin barrier to promote bacterial access to target enterocytes and ultimately toxin delivery, suggesting that EatA plays a crucial role in the molecular pathogenesis of ETEC. These findings may inform novel approaches to prevention of acute diarrheal illness as well as the sequelae associated with ETEC and other pathogens that rely on EatA and similar proteases for efficient interaction with their human hosts.

The IgGFc-binding protein FCGBP is secreted with all GDPH sequences cleaved but maintained by interfragment disulfide bonds.

Mucus forms an important protective barrier that minimizes bacterial contact with the colonic epithelium. Intestinal mucus is organized in a complex network with several specific proteins, including the mucin-2 (MUC2) and the abundant IgGFc-binding protein, FCGBP. FCGBP is expressed in all intestinal goblet cells and is secreted into the mucus. It is comprised of repeated von Willebrand D (vWD) domain assemblies, most of which have a GDPH amino acid sequence that can be autocatalytically cleaved, as previously observed in the mucins MUC2 and mucin-5AC. However, the functions of FCGBP in the mucus are not understood. We show that all vWD domains of FCGBP with a GDPH sequence are cleaved and that these cleavages occur early during biosynthesis in the endoplasmic reticulum. All cleaved fragments, however, remain connected via a disulfide bond within each vWD domain. This cleavage generates a C-terminal-reactive Asp-anhydride that could react with other molecules, such as MUC2, but this was not observed. Quantitative analyses by MS showed that FCGBP was mainly soluble in chaotropic solutions, whereas MUC2 was insoluble, and most of the secreted FCGBP was not covalently bound to MUC2. Although FCGBP has been suggested to bind immunoglobulin G, we were unable to reproduce this binding in vitro using purified proteins. In conclusion, while the function of FCGBP is still unknown, our results suggest that it does not contribute to covalent crosslinking in the mucus, nor incorporate immunoglobulin G into mucus, instead the single disulfide bond linking each fragment could mediate controlled dissociation.

ProteoSushi: A Software Tool to Biologically Annotate and Quantify Modification-Specific, Peptide-Centric Proteomics Data Sets.

Large-scale proteomic profiling of protein post-translational modifications has provided important insights into the regulation of cell signaling and disease. These modification-specific proteomics workflows nearly universally enrich modified peptides prior to mass spectrometry analysis, but protein-centric proteomic software tools have many limitations evaluating and interpreting these peptide-centric data sets. We, therefore, developed ProteoSushi, a software tool tailored to analysis of each modified site in peptide-centric proteomic data sets that is compatible with any post-translational modification or chemical label. ProteoSushi uses a unique approach to assign identified peptides to shared proteins and genes, minimizing redundancy by prioritizing shared assignments based on UniProt annotation score and optional user-supplied protein/gene lists. ProteoSushi simplifies quantitation by summing or averaging intensities for each modified site, merging overlapping peptide charge states, missed cleavages, spectral matches, and variable modifications into a single value. ProteoSushi also annotates each PTM site with the most up-to-date biological information available from UniProt, such as functional roles or known modifications, the protein domain in which the site resides, the protein's subcellular location and function, and more. ProteoSushi has a graphical user interface for ease of use. ProteoSushi's flexibility and combination of analysis features streamlines peptide-centric data processing and knowledge mining of large modification-specific proteomics data sets.

NOX1-dependent redox signaling potentiates colonic stem cell proliferation to adapt to the intestinal microbiota by linking EGFR and TLR activation.

The colon epithelium is a primary point of interaction with the microbiome and is regenerated by a few rapidly cycling colonic stem cells (CSCs). CSC self-renewal and proliferation are regulated by growth factors and the presence of bacteria. However, the molecular link connecting the diverse inputs that maintain CSC homeostasis remains largely unknown. We report that CSC proliferation is mediated by redox-dependent activation of epidermal growth factor receptor (EGFR) signaling via NADPH oxidase 1 (NOX1). NOX1 expression is CSC specific and is restricted to proliferative CSCs. In the absence of NOX1, CSCs fail to generate ROS and have a reduced proliferation rate. NOX1 expression is regulated by Toll-like receptor activation in response to the microbiota and serves to link CSC proliferation with the presence of bacterial components in the crypt. The TLR-NOX1-EGFR axis is therefore a critical redox signaling node in CSCs facilitating the quiescent-proliferation transition and responds to the microbiome to maintain colon homeostasis.

Metaproteomics Analysis of Host-Microbiota Interfaces.

Metaproteomics of host-microbiome interfaces comprises the analysis of complex mixtures of bacteria, archaea, fungi, and viruses in combination with its host cells. Microbial niches can be found all over the host including the skin, oral cavity, and the intestine and are considered to be essential for the homeostasis. The complex interactions between the host and diverse commensal microbiota are poorly characterized while of great interest as dysbiosis is associated with the development of various inflammatory and metabolic diseases. The metaproteomics workflows to study these interfaces are currently being established, and many challenges remain. The major challenge is the large diversity in species composition that make up the microbiota, which results in complex samples that require extended mass spectrometry analysis time. In addition, current database search strategies are not developed to the size of the search space required for unbiased microbial protein identification.Here, we describe a workflow for the proteomics analysis of microbial niches with a focus on intestinal mucus layer. We will cover step-by-step the sample collection, sample preparation, liquid chromatography-mass spectrometry, and data analysis.

ProteoClade: A taxonomic toolkit for multi-species and metaproteomic analysis.

We present ProteoClade, a Python toolkit that performs taxa-specific peptide assignment, protein inference, and quantitation for multi-species proteomics experiments. ProteoClade scales to hundreds of millions of protein sequences, requires minimal computational resources, and is open source, multi-platform, and accessible to non-programmers. We demonstrate its utility for processing quantitative proteomic data derived from patient-derived xenografts and its speed and scalability enable a novel de novo proteomic workflow for complex microbiota samples.

Protein Turnover in Epithelial Cells and Mucus along the Gastrointestinal Tract Is Coordinated by the Spatial Location and Microbiota.

The gastrointestinal tract is covered by a single layer of epithelial cells that, together with the mucus layers, protect the underlying tissue from microbial invasion. The epithelium has one of the highest turnover rates in the body. Using stable isotope labeling, high-resolution mass spectrometry, and computational analysis, we report a comprehensive dataset of the turnover of more than 3,000 and the expression of more than 5,000 intestinal epithelial cell proteins, analyzed under conventional and germ-free conditions across five different segments in mouse intestine. The median protein half-life is shorter in the small intestine than in the colon. Differences in protein turnover rates along the intestinal tract can be explained by distinct physiological and immune-related functions between the small and large intestine. An absence of microbiota results in an approximately 1 day longer protein half-life in germ-free animals.

Proteomic analysis of follicular fluid during human ovulation.

INTRODUCTION: Human ovulation is a biologically complex process that involves several biochemical factors, promoting follicular rupture and release of a fertilizable oocyte. Proteins which are present in follicular fluid at high concentrations during ovulation are likely to be active participants in the biochemical pathways of ovulation. The aim of the study was to identify, by use of a modern proteomic technique, proteins of human follicular fluid which are differentially regulated during ovulation of the natural menstrual cycle. MATERIAL AND METHODS: This prospective experimental study over 3 years included women planned for laparoscopic sterilization. During surgery, retrieval of the dominant follicle was performed either at the preovulatory stage or during ovulation. Four women of preovulatory phase and four women of ovulatory phase met the predetermined criteria of hormone levels for respective phases, and samples of these were finally included out of the 15 women operated. Follicular fluid was aspirated from the excised follicle and subjected to mass spectrometry with the isobaric tags for relative and absolute quantification (iTRAQ) technology for isobaric tagging of peptides. This enables simultaneous identification and quantification of proteins. The protein profiles of the follicular fluid of the preovulatory phase and the ovulatory phase were analyzed, and proteins that were present were identified. RESULTS: A total of 502 proteins were identified, several of which previously have not been identified in human follicular fluid. Of the 115 proteins that were found in all samples, 20 proteins were at higher levels during ovulation. These were inflammatory-related proteins, coagulation factors, proteins in lipid metabolism, complement factors and antioxidants. Five proteins were present in lower levels during ovulation, with three being enzymes and the other two proteins of lipid metabolism and iron transport. CONCLUSIONS: Twenty-five follicular fluid proteins, with differential regulation during ovulation, were identified in human follicular fluid of the natural menstrual cycle. These proteins may have essential roles in the ovulatory cascade.

Spatial and temporal alterations in protein structure by EGF regulate cryptic cysteine oxidation.

Stimulation of plasma membrane receptor tyrosine kinases (RTKs), such as the epidermal growth factor receptor (EGFR), locally increases the abundance of reactive oxygen species (ROS). These ROS then oxidize cysteine residues in proteins to potentiate downstream signaling. Spatial confinement of ROS is an important regulatory mechanism of redox signaling that enables the stimulation of different RTKs to oxidize distinct sets of downstream proteins. To uncover additional mechanisms that specify cysteines that are redox regulated by EGF stimulation, we performed time-resolved quantification of the EGF-dependent oxidation of 4200 cysteine sites in A431 cells. Fifty-one percent of cysteines were statistically significantly oxidized by EGF stimulation. Furthermore, EGF induced three distinct spatiotemporal patterns of cysteine oxidation in functionally organized protein networks, consistent with the spatial confinement model. Unexpectedly, protein crystal structure analysis and molecular dynamics simulations indicated widespread redox regulation of cryptic cysteine residues that are solvent exposed only upon changes in protein conformation. Phosphorylation and increased flux of nucleotide substrates served as two distinct modes by which EGF specified the cryptic cysteine residues that became solvent exposed and redox regulated. Because proteins that are structurally regulated by different RTKs or cellular perturbations are largely unique, these findings suggest that solvent exposure and redox regulation of cryptic cysteine residues contextually delineate redox signaling networks.

The Nlrp6 inflammasome is not required for baseline colonic inner mucus layer formation or function.

The inner mucus layer (IML) is a critical barrier that protects the colonic epithelium from luminal threats and inflammatory bowel disease. Innate immune signaling is thought to regulate IML formation via goblet cell Nlrp6 inflammasome activity that controls secretion of the mucus structural component Muc2. We report that isolated colonic goblet cells express components of several inflammasomes; however, analysis of IML properties in multiple inflammasome-deficient mice, including littermate-controlled Nlrp6-/- , detect a functional IML barrier in all strains. Analysis of mice lacking inflammasome substrate cytokines identifies a defective IML in Il18-/- mice, but this phenotype is ultimately traced to a microbiota-driven, Il18-independent effect. Analysis of phenotypic transfer between IML-deficient and IML-intact mice finds that the Bacteroidales family S24-7 (Muribaculaceae) and genus Adlercrutzia consistently positively covary with IML barrier function. Together, our results demonstrate that baseline IML formation and function is independent of inflammasome activity and highlights the role of the microbiota in determining IML barrier function.

Structural weakening of the colonic mucus barrier is an early event in ulcerative colitis pathogenesis.

OBJECTIVE: The colonic inner mucus layer protects us from pathogens and commensal-induced inflammation, and has been shown to be defective in active UC. The aim of this study was to determine the underlying compositional alterations, their molecular background and potential contribution to UC pathogenesis. DESIGN: In this single-centre case-control study, sigmoid colon biopsies were obtained from patients with UC with ongoing inflammation (n=36) or in remission (n=28), and from 47 patients without colonic disease. Mucus samples were collected from biopsies ex vivo, and their protein composition analysed by nanoliquid chromatography-tandem mass spectrometry. Mucus penetrability and goblet cell responses to microbial stimulus were assessed in a subset of patients. RESULTS: The core mucus proteome was found to consist of a small set of 29 secreted/transmembrane proteins. In active UC, major structural mucus components including the mucin MUC2 (p<0.0001) were reduced, also in non-inflamed segments. Active UC was associated with decreased numbers of sentinel goblet cells and attenuation of the goblet cell secretory response to microbial challenge. Abnormal penetrability of the inner mucus layer was observed in a subset of patients with UC (12/40; 30%). Proteomic alterations in penetrable mucus samples included a reduction of the SLC26A3 apical membrane anion exchanger, which supplies bicarbonate required for colonic mucin barrier formation. CONCLUSION: Core mucus structural components were reduced in active UC. These alterations were associated with attenuation of the goblet cell secretory response to microbial challenge, but occurred independent of local inflammation. Thus, mucus abnormalities are likely to contribute to UC pathogenesis.

Calcium-activated Chloride Channel Regulator 1 (CLCA1) Controls Mucus Expansion in Colon by Proteolytic Activity.

Many epithelial surfaces of the body are covered with protective mucus, and disrupted mucus homeostasis is coupled to diseases such as ulcerative colitis, helminth infection, cystic fibrosis, and chronic obstructive lung disease. However, little is known how a balanced mucus system is maintained. By investigating the involvement of proteases in colonic mucus dynamics we identified metalloprotease activity to be a key contributor to mucus expansion. The effect was mediated by calcium-activated chloride channel regulator 1 (CLCA1) as application of recombinant CLCA1 on intestinal mucus in freshly dissected tissue resulted in increased mucus thickness independently of ion and mucus secretion, but dependent on its metallohydrolase activity. Further, CLCA1 modulated mucus dynamics in both human and mouse, and knock-out of CLCA1 in mice was compensated for by cysteine proteases. Our results suggest that CLCA1 is involved in intestinal mucus homeostasis by facilitating processing and removal of mucus to prevent stagnation. In light of our findings, we suggest future studies to investigate if upregulation of CLCA1 in diseases associated with mucus accumulation could facilitate removal of mucus in an attempt to maintain homeostasis.

Multiple enzyme approach for the characterization of glycan modifications on the C-terminus of the intestinal MUC2mucin.

The polymeric mucin MUC2 constitutes the main structural component of the mucus that covers the colon epithelium. The protein's central mucin domain is highly O-glycosylated and binds water to provide lubrication and prevent dehydration, binds bacteria, and separates the bacteria from the epithelial cells. Glycosylation outside the mucin domain is suggested to be important for proper protein folding and protection against intestinal proteases. However, glycosylation of these regions of the MUC2 has not been extensively studied. A purified 250 kDa recombinant protein containing the last 981 amino acids of human MUC2 was produced in CHO-K1 cells. The protein was analyzed before and after PNGase F treatment, followed by in-gel digestion with trypsin, chymotrypsin, subtilisin, or Asp-N. Peptides were analyzed by nLC/MS/MS using a combination of CID, ETD, and HCD fragmentation. The multiple enzyme approach increased peptide coverage from 36% when only using trypsin, to 86%. Seventeen of the 18 N-glycan consensus sites were identified as glycosylated. Fifty-six N-glycopeptides covering 10 N-glycan sites, and 14 O-glycopeptides were sequenced and characterized. The presented method of protein digestion can be used to gain better insights into the density and complexity of glycosylation of complex glycoproteins such as mucins.

Altered expression of autoimmune regulator in infant down syndrome thymus, a possible contributor to an autoimmune phenotype.

Down syndrome (DS), caused by trisomy of chromosome 21, is associated with immunological dysfunctions such as increased frequency of infections and autoimmune diseases. Patients with DS share clinical features, such as autoimmune manifestations and specific autoantibodies, with patients affected by autoimmune polyendocrine syndrome type 1. Autoimmune polyendocrine syndrome type 1 is caused by mutations in the autoimmune regulator (AIRE) gene, located on chromosome 21, which regulates the expression of tissue-restricted Ags (TRAs) in thymic epithelial cells. We investigated the expression of AIRE and TRAs in DS and control thymic tissue using quantitative PCR. AIRE mRNA levels were elevated in thymic tissue from DS patients, and trends toward increased expression of the AIRE-controlled genes INSULIN and CHRNA1 were found. Immunohistochemical stainings showed altered cell composition and architecture of the thymic medulla in DS individuals with increased frequencies of AIRE-positive medullary epithelial cells and CD11c-positive dendritic cells as well as enlarged Hassall's corpuscles. In addition, we evaluated the proteomic profile of thymic exosomes in DS individuals and controls. DS exosomes carried a broader protein pool and also a larger pool of unique TRAs compared with control exosomes. In conclusion, the increased AIRE gene dose in DS could contribute to an autoimmune phenotype through multiple AIRE-mediated effects on homeostasis and function of thymic epithelial cells that affect thymic selection processes.

The mucus and mucins of the goblet cells and enterocytes provide the first defense line of the gastrointestinal tract and interact with the immune system.

The gastrointestinal tract is covered by mucus that has different properties in the stomach, small intestine, and colon. The large highly glycosylated gel-forming mucins MUC2 and MUC5AC are the major components of the mucus in the intestine and stomach, respectively. In the small intestine, mucus limits the number of bacteria that can reach the epithelium and the Peyer's patches. In the large intestine, the inner mucus layer separates the commensal bacteria from the host epithelium. The outer colonic mucus layer is the natural habitat for the commensal bacteria. The intestinal goblet cells secrete not only the MUC2 mucin but also a number of typical mucus components: CLCA1, FCGBP, AGR2, ZG16, and TFF3. The goblet cells have recently been shown to have a novel gate-keeping role for the presentation of oral antigens to the immune system. Goblet cells deliver small intestinal luminal material to the lamina propria dendritic cells of the tolerogenic CD103(+) type. In addition to the gel-forming mucins, the transmembrane mucins MUC3, MUC12, and MUC17 form the enterocyte glycocalyx that can reach about a micrometer out from the brush border. The MUC17 mucin can shuttle from a surface to an intracellular vesicle localization, suggesting that enterocytes might control and report epithelial microbial challenge. There is communication not only from the epithelial cells to the immune system but also in the opposite direction. One example of this is IL10 that can affect and improve the properties of the inner colonic mucus layer. The mucus and epithelial cells of the gastrointestinal tract are the primary gate keepers and controllers of bacterial interactions with the host immune system, but our understanding of this relationship is still in its infancy.

Membrane protein profiling of human colon reveals distinct regional differences.

The colonic epithelium is a highly dynamic system important for the regulation of ion and water homeostasis via absorption and secretion and for the maintenance of a protective barrier between the outer milieu and the inside of the body. These processes are known to gradually change along the length of the colon, although a complete characterization at the protein level is lacking. We therefore analyzed the membrane proteome of isolated human (n = 4) colonic epithelial cells from biopsies obtained via routine colonoscopy for four segments along the large intestine: ascending, transverse, descending, and sigmoid colon. Label-free quantitative proteomic analyses using high-resolution mass spectrometry were performed on enriched membrane proteins. The results showed a stable level for the majority of membrane proteins but a distinct decrease in proteins associated with bacterial sensing, cation transport, and O-glycosylation in the proximal to distal regions. In contrast, proteins involved in microbial defense and anion transport showed an opposing gradient and increased toward the distal end. The gradient of ion-transporter proteins could be directly related to previously observed ion transport activities. All individual glycosyltransferases required for the O-glycosylation of the major colonic mucin MUC2 were observed and correlated with the known glycosylation variation along the colon axis. This is the first comprehensive quantitative dataset of membrane protein abundance along the human colon and will add to the knowledge of the physiological function of the different regions of the colonic mucosa. Mass spectrometry data have been deposited to the ProteomeXchange with the identifier PXD000987.

Inhibition of Cyclooxygenase-2 Prevents Chronic and Recurrent Cystitis.

The spread of multidrug-resistant microorganisms globally has created an urgent need for novel therapeutic strategies to combat urinary tract infections (UTIs). Immunomodulatory therapy may provide benefit, as treatment of mice with dexamethasone during acute UTI improved outcome by reducing the development of chronic cystitis, which predisposes to recurrent infection. Here we discovered soluble biomarkers engaged in myeloid cell development and chemotaxis that were predictive of future UTI recurrence when elevated in the sera of young women with UTI. Translation of these findings revealed that temperance of the neutrophil response early during UTI, and specifically disruption of bladder epithelial transmigration of neutrophils by inhibition of cyclooxygenase-2, protected mice against chronic and recurrent cystitis. Further, proteomics identified bladder epithelial remodeling consequent to chronic infection that enhances sensitivity to neutrophil damage. Thus, cyclooxygenase-2 expression during acute UTI is a critical molecular trigger determining disease outcome and drugs targeting cyclooxygenase-2 could prevent recurrent UTI.

Characterization of human thymic exosomes.

Exosomes are nanosized membrane-bound vesicles that are released by various cell types and are capable of carrying proteins, lipids and RNAs which can be delivered to recipient cells. Exosomes play a role in intercellular communication and have been described to mediate immunologic information. In this article we report the first isolation and characterization of exosomes from human thymic tissue. Using electron microscopy, particle size determination, density gradient measurement, flow cytometry, proteomic analysis and microRNA profiling we describe the morphology, size, density, protein composition and microRNA content of human thymic exosomes. The thymic exosomes share characteristics with previously described exosomes such as antigen presentation molecules, but they also exhibit thymus specific features regarding surface markers, protein content and microRNA profile. Interestingly, thymic exosomes carry proteins that have a tissue restricted expression in the periphery which may suggest a role in T cell selection and the induction of central tolerance. We speculate that thymic exosomes may provide the means for intercellular information exchange necessary for negative selection and regulatory T cell formation of the developing thymocytes within the human thymic medulla.