Nadine Waldschmitt

Evidence for Estrogen-Dependent Uterine Serpin (SERPINA14) Expression During Estrus in the Bovine Endometrial Glandular Epithelium and Lumen

Uterine secretions have a dominant impact on the environment in which embryo development takes place. The uterine serpins (SERPINA14, previously known as UTMP) are found most abundantly during pregnancy in the uterus of ruminants. Although progesterone is currently assumed to be the major regulator of SERPINA14 expression, our recent study of transcriptome changes in bovine endometrium during the estrous cycle unexpectedly detected a marked upregulation of SERPINA14 mRNA levels at estrus. The present study describes the full-length mRNA sequence, genomic organization, and putative promoter elements of the SERPINA14 gene. The SERPINA14 mRNA abundance was quantified by real-time RT-PCR in intercaruncular endometrium at several time points during the estrous cycle and early pregnancy. Highest levels were found at estrus, followed by a dramatic decrease and a moderate expression during the luteal phase. Transcript levels were higher in pregnant endometrium compared with controls at Day 18. At estrus, immunoreactive protein was localized in deep glandular epithelium, and Western blotting concomitantly showed the 52-kDa form in uterine flushings. SERPINA14 mRNA was significantly upregulated in glandular endometrial cells in vitro after stimulation with estradiol-17beta and progesterone, but not after interferon-tau treatment. Our results clearly demonstrate that SERPINA14 appears distinctly in bovine endometrium during the estrus phase. A supporting role toward providing a well-prepared endometrial environment for passing gametes, especially sperm, is assumed.

Mitochondrial function controls intestinal epithelial stemness and proliferation

Control of intestinal epithelial stemness is crucial for tissue homeostasis. Disturbances in epithelial function are implicated in inflammatory and neoplastic diseases of the gastrointestinal tract. Here we report that mitochondrial function plays a critical role in maintaining intestinal stemness and homeostasis. Using intestinal epithelial cell (IEC)-specific mouse models, we show that loss of HSP60, a mitochondrial chaperone, activates the mitochondrial unfolded protein response (MT-UPR) and results in mitochondrial dysfunction. HSP60-deficient crypts display loss of stemness and cell proliferation, accompanied by epithelial release of WNT10A and RSPO1. Sporadic failure of Cre-mediated Hsp60 deletion gives rise to hyperproliferative crypt foci originating from OLFM4+ stem cells. These effects are independent of the MT-UPR-associated transcription factor CHOP. In conclusion, compensatory hyperproliferation of HSP60+ escaper stem cells suggests paracrine release of WNT-related factors from HSP60-deficient, functionally impaired IEC to be pivotal in the control of the proliferative capacity of the stem cell niche.

Catechols in caffeic acid phenethyl ester are essential for inhibition of TNF-mediated IP-10 expression through NF-κB-dependent but HO-1- and p38-independent mechanisms in mouse intestinal epithelial cells

SCOPE Caffeic acid phenethyl ester (CAPE) is an active constituent of honeybee propolis inhibiting nuclear factor (NF)-κB. The aims of our study were to provide new data on the functional relevance and mechanisms underlying the role of CAPE in regulating inflammatory processes at the epithelial interface in the gut and to determine the structure/activity relationship of CAPE. METHODS AND RESULTS CAPE significantly inhibited TNF-induced IP-10 expression in intestinal epithelial cells. Using various analogues, we demonstrated that substitution of catechol hydroxyl groups and addition of one extra hydroxyl group on ring B reversed the functional activity of CAPE to inhibit IP-10 production. The anti-inflammatory potential of CAPE was confirmed in ileal tissue explants and embryonic fibroblasts derived from TNF(ΔARE/+) mice. Interestingly, CAPE inhibited both TNF- and LPS-induced IP-10 production in a dose-dependent manner, independently of p38 MAPK, HO-1 and Nrf2 signaling pathways. We found that CAPE did not inhibit TNF-induced IκB phosphorylation/degradation or nuclear translocation of RelA/p65, but targeted downstream signaling events at the level of transcription factor recruitment to the gene promoter. CONCLUSION This study reveals the structure-activity effects and anti-inflammatory potential of CAPE in the intestinal epithelium.

C/EBP homologous protein inhibits tissue repair in response to gut injury and is inversely regulated with chronic inflammation.

Loss of intestinal epithelial cell (IEC) homeostasis and apoptosis negatively affect intestinal barrier function. Uncontrolled activation of the unfolded protein response (UPR) in IEC contributes to an impaired barrier and is implicated in the pathogenesis of inflammatory bowel diseases. However, the contribution of the UPR target gene C/EBP homologous protein (CHOP), an apoptosis-associated transcription factor, to inflammation-related disease susceptibility remains unclear. Consistent with observations in patients with ulcerative colitis, we show that despite UPR activation in the epithelium, CHOP expression was reduced in mouse models of T-cell-mediated and bacteria-driven colitis. To elucidate the molecular mechanisms of IEC-specific CHOP expression, we generated a conditional transgenic mouse model (ChopIEC Tg/Tg). Chop overexpression increased the susceptibility toward dextran sodium sulfate (DSS)-induced intestinal inflammation and mucosal tissue injury. Furthermore, a delayed recovery from DSS-induced colitis and impaired closure of mechanically induced mucosal wounds was observed. Interestingly, these findings seemed to be independent of CHOP-mediated apoptosis. In vitro and in vivo cell cycle analyses rather indicated a role for CHOP in epithelial cell proliferation. In conclusion, these data show that IEC-specific overexpression impairs epithelial cell proliferation and mucosal tissue regeneration, suggesting an important role for CHOP beyond mediating apoptosis.

Activated ATF6 Induces Intestinal Dysbiosis and Innate Immune Response to Promote Colorectal Tumorigenesis

BACKGROUND & AIMS Activating transcription factor 6 (ATF6) regulates endoplasmic reticulum stress. We studied whether ATF6 contributes to the development of colorectal cancer (CRC) using tissue from patients and transgenic mice. METHODS We analyzed data from 541 patients with CRC in The Cancer Genome Atlas database for genetic variants and aberrant expression levels of unfolded protein response genes. Findings were validated in a cohort of 83 patients with CRC in Germany. We generated mice with intestinal epithelial cell-specific expression of the active form of Atf6 (nATF6IEC) from 2 alleles (homozygous), mice with expression of nATF6IEC from 1 allele (heterozygous), and nATF6IECfl/fl mice (controls). All nATF6IEC mice were housed under either specific-pathogen-free or germ-free conditions. Cecal microbiota from homozygous nATF6IEC mice or control mice was transferred into homozygous nATF6IEC mice or control mice. nATF6IEC mice were crossed with mice with disruptions in the myeloid differentiation primary response gene 88 and toll-like receptor adaptor molecule 1 gene (Myd88/Trif-knockout mice). Intestinal tissues were collected from mice and analyzed by histology, immunohistochemistry, immunoblots, gene expression profiling of unfolded protein response and inflammatory genes, array-based comparative genome hybridization, and 16S ribosomal RNA gene sequencing. RESULTS Increased expression of ATF6 was associated with reduced disease-free survival times of patients with CRC. Homozygous nATF6IEC mice developed spontaneous colon adenomas at 12 weeks of age. Compared with controls, homozygous nATF6IEC mice had changes in the profile of their cecal microbiota, increased proliferation of intestinal epithelial cells, and loss of the mucus barrier-all preceding tumor formation. These mice had increased penetration of bacteria into the inner mucus layer and activation of signal transducer and activator of transcription 3, yet inflammation was not observed at the pretumor or tumor stages. Administration of antibiotics to homozygous nATF6IEC mice greatly reduced tumor incidence, and germ-free housing completely prevented tumorigenesis. Analysis of nATF6IEC MyD88/TRIF-knockout mice showed that tumor initiation and growth required MyD88/TRIF-dependent activation of signal transducer and activator of transcription 3. Transplantation of cecal microbiota from nATF6IEC mice and control mice, collected before tumor formation, caused tumor formation in ex-germ-free nATF6IEC mice. CONCLUSIONS In patients with CRC, ATF6 was associated with reduced time of disease-free survival. In studies of nATF6IEC mice, we found sustained intestinal activation of ATF6 in the colon to promote dysbiosis and microbiota-dependent tumorigenesis.

Role of Incretin Hormones in Bowel Diseases

Enteroendocrine cells (EEC) have been studied extensively for their ability to regulate gastrointestinal motility and insulin release by secretion of peptide hormones. In particular, the L cell-derived incretin glucagon-like peptide 1 has gained enormous attention due to its insulinotropic action and relevance in the treatment of type 2 diabetes. Yet, accumulating data indicates a critical role for EEC and incretins in metabolic adaptation and in orchestrating immune responses beyond blood glucose control. EEC actively sense the lamina propria and luminal environment including the microbiota via receptors and transporters, subsequently mediating signals by secreting hormones and cytokines. Data indicate that immune cells and cytokine-mediated signaling impacts EEC numbers and function during infection and chronic inflammation of the gut, suggesting EEC not only to play a role in these pathologies but also being a target of inflammatory processes. This review presents data on the interrelation of incretins and inflammatory signaling. It focuses on the impact of intestinal inflammation, in particular inflammatory bowel disease, on EEC and the potential role of EEC and incretins in these pathologies. Furthermore, it highlights endoplasmic reticulum unfolded protein response, cytokines and the intestinal microbiota as possible targets of inflammatory and EEC signaling.

Microbiome and Diseases: Inflammatory Bowel Diseases

Inflammatory bowel diseases, such as Crohn’s disease and ulcerative colitis, are chronically relapsing, immune-mediated disorders of the gastrointestinal tract that have been steadily increasing over the past decades. The hallmark of IBD is an uncontrolled manifestation of intestinal and extraintestinal inflammation within genetically susceptible individuals. Herein, compelling research on host genetics pave the way to a better understanding of disease pathogenesis. Over 200 genetic risk factors have been identified showing a disturbed cross talk of the immune epithelial cell microbiota axis. Additionally, epidemiologic studies pointed toward Western lifestyle and habits as part of central environmental factors contributing to both development and maintenance of intestinal inflammation. In this regard, the gut microbiota is thought to play a decisive role in disease progression. The intestinal microbiota was unequivocally shown to be indispensable in orchestrating the development and functionality of the immune system further having a critical impact on both intestinal homeostasis and inflammation in preclinical models. Even though profound changes in the composition of the intestinal microbiota have been frequently observed in human IBD, unraveling cause and consequences of intestinal dysbiosis need further understanding of the interaction between host genetics, microbial ecosystems, and environmental triggers. In this chapter we will discuss different aspects of the etiology of intestinal inflammation and particularly address the role of host-microbe interaction in disease development, progression, and intervention.

Microbial Signatures as a Predictive Tool in IBD—Pearls and Pitfalls

Studies of microbial signatures have improved our understanding of the role of dysbiosis in gut microbiota for the pathogenesis of inflammatory bowel disease (IBD). New technological advances such as next-generation sequencing facilitate investigations on large patient cohorts, but require methodological considerations regarding study design, sample processing, data analysis, and integration. Here, we summarize recent study approaches in microbial ecology with respect to IBD research and discuss crucial process steps for the production and integration of adequate data sets.

Tu1760 Functional Evidence for a Causative Role of the Intestinal Microbiota in Crohn's Disease-Like Ileitis Using Antibiotic-Treated and Germ-Free TNFDeltaare/+ Mice

BACKGROUND: An increased prevalence of phylogenetic B2 group Escherichia coli (E. coli) has been associated with active disease in IBD patients (IBDEC). Many of the B2 group strains harbor virulence genes present in extraintestinal pathogenic E.coli including hemolysin (hly) and cytotoxic necrotizing factor type 1 (CNF1) the loci of which is often linked. Here we investigated the potential of B2 strains of E.coli isolated from IBD patients to damage the integrity of the intestinal epithelium and the potential contribution of Hly and CNF1. METHODS: E coli strains isolated from 35 patients with IBD were tested for virulence (Petersen et al 2009) and of those isolate C691-04A showed high cytotoxicity on dendritic cells ( submitted) and a rapidly decrease of the trans-epithelial electric resistance (TEER) of a monolayer of intestinal epithelial cell lines. Genome sequencing of C691-04A confirmed the presence of hly cluster. It is known that some E coli have two hly-clusters. In order to investigate the role of hly and CNF1 in disruption of the epithelial integrity the following mutants were created in strain C691-04A; 1: CAS965, hly cluster II upstream of CNF1; 2: CAS954, hly cluster I mutant; 3: CAS956 is a knock out of both loci of hly; 4: CAS964, CNF1 mutant; 5: CAS966, clbA mutant. Caco-2 cells were grown to confluence in 24-well trans-well devices for 14 days to form a polarized cell monolayer and TER measured constantly during exposure to E. coli strains at MOI 10. The wild type and commensal E. coli were included as controls. RESULTS: E. coli C691-04A WT as well as CNF1, clbA and single loci of hly mutants from cluster I and II were all able to damage Caco-2 cell tight junctions in less than 6 hrs co-culture (see figure). Confocal microscopy of occludin stained trans-well sections revealed loss of occludin from the tight junction within 3 hours. However, this phenotype was lost in a mutant with knock out of both hly loci (p<0.001). Commensal E. coli isolated from healthy controls also did not cause loss of TER in 6 h. CONCLUSION: IBD-associated E. coli such as C691-04A can cause rapid loss of tight junction integrity in differentiated Caco-2 cell monolayers. This effect is abolished in a mutant with knock out of both loci of the hly genes and diminished in one of the single knockout mutants. These results suggest that high Hly expression may be a mechanism by which specific strains of E. coli pathobionts can contribute to epithelial barrier dysfunction and pathophysiology of disease in IBD.