Different Forms of TFF3 in the Human Endocervix, including a Complex with IgG Fc Binding Protein (FCGBP), and Further Aspects of the Cervico-Vaginal Innate Immune Barrier.

TFF3 is a typical secretory poplypeptide of mucous epithelia belonging to the trefoil factor family (TFF) of lectins. In the intestine, respiratory tract, and saliva, TFF3 mainly exists as a high-molecular-mass complex with IgG Fc binding protein (FCGBP), which is indicative of a role in mucosal innate immunity. For the first time, we identified different forms of TFF3 in the endocervix, i.e., monomeric and homodimeric TFF3, as well as a high-molecular-mass TFF3-FCGBP complex; the latter also exists in a hardly soluble form. Immunohistochemistry co-localized TFF3 and FCGBP. Expression analyses of endocervical and post-menopausal vaginal specimens revealed a lack of mucin and TFF3 transcripts in the vaginal specimens. In contrast, genes encoding other typical components of the innate immune defense were expressed in both the endocervix and vagina. Of note, FCGBP is possibly fucosylated. Endocervical specimens from transgender individuals after hormonal therapy showed diminished expression, particularly of FCGBP. Furthermore, mucus swabs from the endocervix and vagina were analyzed concerning TFF3, FCGBP, and lysozyme. It was the aim of this study to illuminate several aspects of the cervico-vaginal innate immune barrier, which is clinically relevant as bacterial and viral infections are also linked to infertility, pre-term birth and cervical cancer.

Expression Profiling along the Murine Intestine: Different Mucosal Protection Systems and Alterations in Tff1-Deficient Animals.

Tff1 is a typical gastric peptide secreted together with the mucin, Muc5ac. Tff1-deficient (Tff1KO) mice are well known for their prominent gastric phenotype and represent a recognized model for antral tumorigenesis. Notably, intestinal abnormalities have also been reported in the past in these animals. Here, we have compared the expression of selected genes in Tff1KO mice and their corresponding wild-type littermates (RT-PCR analyses), focusing on different mucosal protection systems along the murine intestine. As hallmarks, genes were identified with maximum expression in the proximal colon and/or the duodenum: Agr2, Muc6/A4gnt/Tff2, Tff1, Fut2, Gkn2, Gkn3, Duox2/Lpo, Nox1. This is indicative of different protection systems such as Tff2/Muc6, Tff1-Fcgbp, gastrokines, fucosylation, and reactive oxygen species (ROS) in the proximal colon and/or duodenum. Few significant transcriptional changes were observed in the intestine of Tff1KO mice when compared with wild-type littermates, Clca1 (Gob5), Gkn1, Gkn2, Nox1, Tff2. We also analyzed the expression of Tff1, Tff2, and Tff3 in the pancreas, liver, and lung of Tff1KO and wild-type animals, indicating a cross-regulation of Tff gene expression. Furthermore, on the protein level, heteromeric Tff1-Fcgbp and various monomeric Tff1 forms were identified in the duodenum and a high-molecular-mass Tff2/Muc6 complex was identified in the proximal colon (FPLC, proteomics).

The Forms of the Lectin Tff2 Differ in the Murine Stomach and Pancreas: Indications for Different Molecular Functions.

The lectin TFF2 belongs to the trefoil factor family (TFF). This polypeptide is typically co-secreted with the mucin MUC6 from gastric mucous neck cells, antral gland cells, and duodenal Brunner glands. Here, TFF2 fulfills a protective function by forming a high-molecular-mass complex with the MUC6, physically stabilizing the mucus barrier. In pigs and mice, and slightly in humans, TFF2 is also synthesized in the pancreas. Here, we investigated the murine stomach, pancreas, and duodenum by fast protein liquid chromatography (FPLC) and proteomics and identified different forms of Tff2. In both the stomach and duodenum, the predominant form is a high-molecular-mass complex with Muc6, whereas, in the pancreas, only low-molecular-mass monomeric Tff2 was detectable. We also investigated the expression of Tff2 and other selected genes in the stomach, pancreas, and the proximal, medial, and distal duodenum (RT-PCR analysis). The absence of the Tff2/Muc6 complex in the pancreas is due to a lack of Muc6. Based on its known motogenic, anti-apoptotic, and anti-inflammatory effects, we propose a protective receptor-mediated function of monomeric Tff2 for the pancreatic ductal epithelium. This view is supported by a report that a loss of Tff2 promotes the formation of pancreatic intraductal mucinous neoplasms.

Profiling of the Bacterial Microbiota along the Murine Alimentary Tract.

Here, the spatial distribution of the bacterial flora along the murine alimentary tract was evaluated using high throughput sequencing in wild-type and Tff3-deficient (Tff3KO) animals. Loss of Tff3 was linked to increased dextran sodium sulfate-induced colitis. This systematic study shows the results of 13 different regions from the esophagus to the rectum. The number of bacterial species (richness) increased from the esophagus to the rectum, from 50 to 200, respectively. Additionally, the bacterial community structure changed continuously; the highest changes were between the upper/middle and lower gastrointestinal compartments when comparing adjacent regions. Lactobacillus was the major colonizer in the upper/middle gastrointestinal tract, especially in the esophagus and stomach. From the caecum, a drastic diminution of Lactobacillus occurred, while members of Lachnospiraceae significantly increased. A significant change occurred in the bacterial community between the ascending and the transverse colon with Bacteroidetes being the major colonizers with relative constant abundance until the rectum. Interestingly, wild-type and Tff3KO animals did not show significant differences in their bacterial communities, suggesting that Tff3 is not involved in alterations of intraluminal or adhesive microbiota but is obviously important for mucosal protection, e.g., of the sensitive stem cells in the colonic crypts probably by a mucus plume.

Molecular Alterations in the Stomach of Tff1-Deficient Mice: Early Steps in Antral Carcinogenesis.

TFF1 is a peptide of the gastric mucosa co-secreted with the mucin MUC5AC. It plays a key role in gastric mucosal protection and repair. Tff1-deficient (Tff1KO) mice obligatorily develop antropyloric adenoma and about 30% progress to carcinomas. Thus, these mice represent a model for gastric tumorigenesis. Here, we compared the expression of selected genes in Tff1KO mice and the corresponding wild-type animals (RT-PCR analyses). Furthermore, we systematically investigated the different molecular forms of Tff1 and its heterodimer partner gastrokine-2 (Gkn2) in the stomach (Western blot analyses). As a hallmark, a large portion of murine Tff1 occurs in a monomeric form. This is unexpected because of its odd number of seven cysteine residues. Probably the three conserved acid amino acid residues (EEE) flanking the 7th cysteine residue allow monomeric secretion. As a consequence, the free thiol of monomeric Tff1 could have a protective scavenger function, e.g., for reactive oxygen/nitrogen species. Furthermore, a minor subset of Tff1 forms a disulfide-linked heterodimer with IgG Fc binding protein (Fcgbp). Of special note, in Tff1KO animals a homodimeric form of Gkn2 was observed. In addition, Tff1KO animals showed strongly reduced Tff2 transcript and protein levels, which might explain their increased sensitivity to Helicobacter pylori infection.

Transcriptional Responses in the Murine Spleen after Toxoplasma gondii Infection: Inflammasome and Mucus-Associated Genes.

The spleen plays an important role in coordinating both adaptive and innate immune responses. Here, the transcriptional response to T. gondii infection in the murine spleen was characterized concerning inflammasome sensors (two different models: seven days after oral or four weeks after intraperitoneal infection). Additionally, Tff1KO and Tff3KO mice were investigated because TFF genes are often upregulated during inflammation. The expression of the pattern-recognition receptors Nlrp3, Nlrp12, and Nlrp1a was significantly increased after infection. This increase was diminished in Tff1KO and Tff3KO mice pointing towards a positive regulation of the inflammatory response by Tff1 and Tff3. Furthermore, the transcription of Tff1 (encoding a motogenic lectin) and other secretory genes was analyzed, i.e., gastrokines (Gkn), IgG Fc binding protein (Fcgbp), and the mucin Muc2. The corresponding gene products belong to an interactome protecting mucous epithelia. Tff1 was significantly induced after infection, which might increase the motility of immune cells. In contrast, Gkn3, Fcgbp, and Muc2 were downregulated seven days after oral infection; whereas four weeks after i.p. infection only Gkn3 remained downregulated. This might be an indication that Gkn3, Fcgbp, and Muc2 are involved in the transient disruption of the splenic architecture and its reorganization, which is characteristic after T. gondii infection.

Increased Cerebral Tff1 Expression in Two Murine Models of Neuroinflammation.

BACKGROUND/AIMS: The trefoil factor family (TFF) peptide TFF1 is a typical secretory product of the gastric mucosa and a very low level of expression occurs in nearly all regions of the murine brain. TFF1 possesses a lectin activity and binding to a plethora of transmembrane glycoproteins could explain the diverse biological effects of TFF1 (e.g., anti-apoptotic effect). It was the aim to test whether TFF expression is changed during neuroinflammation. METHODS: Expression profiling was performed using semi-quantitative RT-PCR analyses in two murine models of neuroinflammation, i.e. Toxoplasma gondii-induced encephalitis and experimental autoimmune encephalomyelitis (EAE), the latter being the most common animal model of multiple sclerosis. Tff1 expression was also localized using RNA in situ hybridization histochemistry. RESULTS: We report for the first time on a significant transcriptional induction in cerebral Tff1 expression in both T. gondii-induced encephalitis and EAE. In contrast, Tff2 and Tff3 expression were not altered. Tff1 transcripts were predominantly localized in the internal granular layer of the cerebellum indicating neuronal expression. Furthermore, also glial cells are expected to express Tff1. Characterization of both experimental models by expression profiling (e.g., inflammasome sensors, inflammatory cytokines, microglial marker Iba1, ependymin related protein 1) revealed differences concerning the expression of the inflammasome sensor Nlrp1 and interleukin 17a. CONCLUSION: The up-regulated expression of Tff1 is probably the result of a complex inflammatory process as its expression is induced by tumor necrosis factor α as well as interleukins 1ß and 17. However on the transcript level, Tff1KO mice did not show any significant signs of an altered immune response after infection with T. gondii in comparison with the wild type animals.

Tff3 is expressed in neurons and microglial cells.

BACKGROUND/AIMS: The trefoil factor family (TFF) peptide TFF3 is typically secreted by mucous epithelia, but is also expressed in the immune system and the brain. It was the aim of this study to determine the cerebral cell types which express Tff3. METHODS: Primary cultures from rat embryonic or neonatal cerebral cortex and hippocampus, respectively, were studied by means of RT-PCR and immunofluorescence. Moreover, Tff3 expression was localized by immunocytochemistry in sections of adult rat cerebellum. RESULTS: Tff3 transcripts were detectable in neural cultures of both the cortex and the hippocampus as well as in glial cell-enriched cultures. Tff3 peptide co-localized with Map2 indicating an expression in neurons in vitro. The neuronal expression was confirmed by immunofluorescence studies of adult rat cerebellum. Furthermore, Tff3 peptide showed also a clear co-localization with Iba-1 in vitro typical of activated microglial cells. CONCLUSION: The neuronal expression of Tff3 is in line with a function of a typical neuropeptide influencing, e.g., fear, memory, depression and motoric skills. The expression in activated microglial cells, which is demonstrated here for the first time, points towards a possible function for Tff3 in immune reactions in the CNS. This opens a plethora of additional possible functions for Tff3 including synaptic plasticity and cognition as well as during neuroinflammatory diseases and psychiatric disorders.

Modulation of cell-cell contacts during intestinal restitution in vitro and effects of epidermal growth factor (EGF).

Mucous epithelia represent a major barrier to the outside world and are capable of undergoing rapid repair after injury by cell migration, a process called "restitution". Here, a sensitive RT-PCR method was applied allowing systematic gene expression analysis of separated stationary and migratory non-transformed IEC-18 and IEC-6 cells after scratch wounding. The focus was on genes related to cell-cell contacts. Furthermore, the effect of epidermal growth factor (EGF) on gene expression was studied. Most of the genes investigated here were down-regulated in migratory cells. Many of the alterations are expected to affect the permeability of tight junctions. Also the nectin-afadin complex of adherens junctions was modulated as well as the expression of both the chemokine receptor CXCR4 and the EGF receptor. Of note, restitution was not accompanied by the epithelial-mesenchymal transition (EMT). EGF treatment severely affected the expression of genes important for cell-cell contact and cell communication such as selected tight junction components, CXCR4, and TFF3. Many of these genes are known to be involved in EMT and metastasis. Of special note, most of the expression changes induced by EGF are in contrast to the changes observed in migratory cells.

Expression profiling of stationary and migratory intestinal epithelial cells after in vitro wounding: restitution is accompanied by cell differentiation.

Rapid repair by cell migration, a process called "restitution", is essential for wound healing of mucous epithelia. Here, an established in vitro model for restitution, i.e., migration of the non-transformed intestinal epithelial cell line IEC-18 after scratch wounding, was investigated. This cell line is also known for its retained differentiation potential. The aim of this study was to test by expression profiling whether the differentiation state is altered during restitution in vitro. Using a sensitive RT-PCR method a systematic analysis of separated stationary and migratory cells was performed 48 h after in vitro wounding. Most characteristically, the differentiation state was changed in migratory cells when compared with stationary cells. For example, migratory cells lost markers of terminal differentiation and changed to a phenotype that assists the process of restitution by up-regulating the expression of genes such as plasminogen activator inhibitor-1, transforming growth factor alpha, heparin-binding EGF-like growth factor, alpha-smooth muscle actin, ornithine decarboxylase, and glyceraldehyde-3-phosphate dehydrogenase. However, there were no unequivocal signs of epithelial-mesenchymal transition (EMT) found in migratory cells.

TFF Peptides Play a Role in the Immune Response Following Oral Infection of Mice with Toxoplasma Gondii.

The peptide trefoil factor family 3 (TFF3) is a major constituent of the intestinal mucus, playing an important role in the repair of epithelial surfaces. To further understand the role of TFF3 in the protection of intestinal epithelium, we tested the influence of TFF3 in a murine Toxoplasma gondii-induced ileitis model. Surprisingly, TFF3(KO) mice showed a reduced immune response in the ileum when compared to wild-type animals. Interleukin-12 and interferon-γ expression levels as well as the number of CD4(+) lymphocytes were reduced in the infected TFF3(KO) mice. These effects were in line with the trend of elevated parasite levels in the ileum. Moreover, TFF1 expression was upregulated in the spleen of infected mice. These initial results indicate that TFF3 is involved in the immune pathology of T. gondii infection-induced intestinal inflammation. Thus far, the mechanisms of how TFF3 influences the immune response are not fully understood. Further studies should identify if TFF3 affects mucus sensing of dendritic cells and how TFF3 is involved in regulating the immune response as an intrinsic secretory peptide of immune cells.