Quercetin Exposure Suppresses the Inflammatory Pathway in Intestinal Organoids from Winnie Mice.

Inflammatory bowel diseases (IBDs) are chronic and relapsing immune disorders that result, or possibly originate, from epithelial barrier defects. Intestinal organoids are a new reliable tool to investigate epithelial response in models of chronic inflammation. We produced organoids from the ulcerative colitis murine model Winnie to explore if the chronic inflammatory features observed in the parental intestine were preserved by the organoids. Furthermore, we investigated if quercetin administration to in vitro cultured organoids could suppress LPS-induced inflammation in wild-type organoids (WT-organoids) and spontaneous inflammation in ulcerative colitis organoids (UC-organoids). Our data demonstrate that small intestinal organoids obtained from Winnie mice retain the chronic intestinal inflammatory features characteristic of the parental tissue. Quercetin administration was able to suppress inflammation both in UC-organoids and in LPS-treated WT-organoids. Altogether, our data demonstrate that UC-organoids are a reliable experimental system for investigating chronic intestinal inflammation and pharmacological responses.

WNT2B Deficiency Causes Enhanced Susceptibility to Colitis Due to Increased Inflammatory Cytokine Production.

BACKGROUND & AIMS: Humans with WNT2B deficiency have severe intestinal disease, including significant inflammatory injury, highlighting a critical role for WNT2B. We sought to understand how WNT2B contributes to intestinal homeostasis. METHODS: We investigated the intestinal health of Wnt2b knock out (KO) mice. We assessed the baseline histology and health of the small intestine and colon, and the impact of inflammatory challenge using dextran sodium sulfate (DSS). We also evaluated human intestinal tissue. RESULTS: Mice with WNT2B deficiency had normal baseline histology but enhanced susceptibility to DSS colitis because of an increased early injury response. Although intestinal stem cells markers were decreased, epithelial proliferation was similar to control subjects. Wnt2b KO mice showed an enhanced inflammatory signature after DSS treatment. Wnt2b KO colon and human WNT2B-deficient organoids had increased levels of CXCR4 and IL6, and biopsy tissue from humans showed increased neutrophils. CONCLUSIONS: WNT2B is important for regulation of inflammation in the intestine. Absence of WNT2B leads to increased expression of inflammatory cytokines and increased susceptibility to gastrointestinal inflammation, particularly in the colon.

M1 and M2 Macrophages Differentially Regulate Colonic Crypt Renewal.

BACKGROUND: The colonic epithelium is the most rapidly renewing tissue in the body and is organized into a single cell layer of invaginations called crypts. Crypt renewal occurs through Lgr5 + gut stem cells situated at the crypt base, which divide, produce daughter cells that proliferate, migrate, differentiate into all the cells required for normal gut function, and are finally shed into the crypt lumen. In health, this rapid renewal helps maintain barrier function next to the hostile gut microbial luminal environment. Inflammation results in an influx of immune cells including inflammatory M1 macrophages into the gut mucosa next to the crypt epithelium, but the direct effect of macrophages on crypt regeneration and renewal are poorly understood. METHODS: Using an in vitro macrophage-crypt coculture model, we show that homeostatic M2 macrophages and inflammatory M1 macrophages confer different effects on the crypt epithelium. RESULTS: Both M1 and M2 increase crypt cell proliferation, with M2 macrophages requiring physical contact with the crypt epithelium, whereas M1 macrophages exert their effect through a secreted factor. Only M1 macrophages reduce goblet and Tuft cell numbers and increase Lgr5 + crypt stem cell numbers, all dependent on physical contact with the crypt epithelium. Further studies showed that M1 macrophages increase the Wnt signaling pathways cyclin D1 and LEF1 through physical contact rather than a secreted factor. CONCLUSIONS: These findings highlight the importance of understanding distinct cellular interactions and direct dialogue between cells and increase our understanding of the contribution of different immune cell subtypes on crypt cell biology during inflammation.

Inflammatory macrophages but not homeostatic macrophages modulate crypt epithelial cell differentiation. Direct physical contact between an inflammatory macrophage and the crypt epithelium is required for regulation of differentiation, but crypt proliferation is via a secreted factor.

HGF/c-Met/ß1-integrin signalling axis induces tunneling nanotubes in A549 lung adenocarcinoma cells.

Tunneling nanotubes (TNTs) are thin cytoplasmic extensions involved in long-distance intercellular communication and can transport intracellular organelles and signalling molecules. In cancer cells, TNT formation contributes to cell survival, chemoresistance, and malignancy. However, the molecular mechanisms underlying TNT formation are not well defined, especially in different cancers. TNTs are present in non-small cell lung cancer (NSCLC) patients with adenocarcinoma. In NSCLC, hepatocyte growth factor (HGF) and its receptor, c-Met, are mutationally upregulated, causing increased cancer cell growth, survival, and invasion. This study identifies c-Met, ß1-integrin, and paxillin as novel components of TNTs in A549 lung adenocarcinoma cells, with paxillin localised at the protrusion site of TNTs. The HGF-induced TNTs in our study demonstrate the ability to transport lipid vesicles and mitochondria. HGF-induced TNT formation is mediated by c-Met and ß1-integrin in conjunction with paxillin, followed by downstream activation of MAPK and PI3K pathways and the Arp2/3 complex. These findings demonstrate a potential novel approach to inhibit TNT formation through targeting HGF/c-Met receptor and ß1-integrin signalling interactions, which has implications for multi-drug targeting in NSCLC.

WNT2B Deficiency Causes Increased Susceptibility to Colitis in Mice and Impairs Intestinal Epithelial Development in Humans.

Background and aims: WNT2B is a canonical Wnt ligand previously thought to be fully redundant with other Wnts in the intestinal epithelium. However, humans with WNT2B deficiency have severe intestinal disease, highlighting a critical role for WNT2B. We sought to understand how WNT2B contributes to intestinal homeostasis. Methods: We investigated the intestinal health of Wnt2b knock out (KO) mice. We assessed the impact of inflammatory challenge to the small intestine, using anti-CD3χ antibody, and to the colon, using dextran sodium sulfate (DSS). In addition, we generated human intestinal organoids (HIOs) from WNT2B-deficient human iPSCs for transcriptional and histological analyses. Results: Mice with WNT2B deficiency had significantly decreased Lgr5 expression in the small intestine and profoundly decreased expression in the colon, but normal baseline histology. The small intestinal response to anti-CD3χ antibody was similar in Wnt2b KO and wild type (WT) mice. In contrast, the colonic response to DSS in Wnt2b KO mice showed an accelerated rate of injury, featuring earlier immune cell infiltration and loss of differentiated epithelium compared to WT. WNT2B-deficient HIOs showed abnormal epithelial organization and an increased mesenchymal gene signature. Conclusion: WNT2B contributes to maintenance of the intestinal stem cell pool in mice and humans. WNT2B deficient mice, which do not have a developmental phenotype, show increased susceptibility to colonic injury but not small intestinal injury, potentially due to a higher reliance on WNT2B in the colon compared to the small intestine.WNT2B deficiency causes a developmental phenotype in human intestine with HIOs showing a decrease in their mesenchymal component and WNT2B-deficient patients showing epithelial disorganization. Data Transparency Statement: All RNA-Seq data will be available through online repository as indicated in Transcript profiling. Any other data will be made available upon request by emailing the study authors.

A highly photostable and versatile two-photon fluorescent probe for the detection of a wide range of intracellular nitric oxide concentrations in macrophages and endothelial cells.

Nitric oxide (NO) is involved in many biological processes affecting the cardiovascular, nervous and immune systems. Intracellular NO can be monitored using fluorescent probes in combination with fluorescence imaging techniques. Most of the currently available NO fluorescent molecular probes are excited via one-photon excitation using UV or Vis light, which results in poor penetration and high photodamage to living tissues. Here, we report a two-photon fluorescent molecular probe, DANPY-NO, able to detect NO in live cells. The probe consists of an o-phenylenediamine linked to a naphthalimide core; and operates via photoinduced electron transfer. DANPY-NO exhibits good sensitivity (LOD of 77.8 nM) and high selectivity towards NO, and is stable over a broad range of pHs. The probe targeted acidic organelles within macrophages and endothelial cells, and demonstrated enhanced photostability over a commercially available NO probe. DANPY-NO was used to selectively detect endogenous NO in RAW264.7ϒ NO- macrophages, THP-1 human leukemic cells, primary mouse (bone marrow-derived) macrophages and endothelial cells. The probe was also able to detect exogenous NO in endothelial cells and distinguish between increasing concentrations of NO. The NO detection was evidenced using confocal laser scanning and two-photon microscopies, and flow cytometry. Further evidence was obtained by recording the changes in the intracellular fluorescence emission spectrum of the probe. Importantly, the probe displayed negligible toxicity to the analysed biological samples. The excellent sensitivity, selectivity, stability and versatility of DANPY-NO confirm its potential for in vitro and in vivo imaging of NO.

A Specific Mutation in Muc2 Determines Early Dysbiosis in Colitis-Prone Winnie Mice.

BACKGROUND: Inflammatory bowel disease (IBD), including Crohn disease (CD) and ulcerative colitis (UC), is a multifactorial disorder characterized by chronic inflammation and altered gut barrier function. Dysbiosis, a condition defined by dysregulation of the gut microbiome, has been reported in patients with IBD and in experimental models of colitis. Although several factors have been implicated in directly affecting gut microbial composition, the genetic determinants impacting intestinal dysbiosis in IBD remain relatively unknown. METHODS: We compared the microbiome of normal, uninflamed wild-type (WT) mice with that of a murine model of UC (ie, Winnie strain). Winnie mice possess a missense mutation in Muc2 that manifests in altered mucus production as early as 4 weeks of age, with ensuing colonic inflammation. To better address the potential role of mutant Muc2 in promoting dysbiosis in Winnie mice, we evaluated homozygous mutant mice (Winnie-/-) with their WT littermates that, after weaning from common mothers, were caged separately according to genotype. Histologic and inflammatory status were assessed over time, along with changes in their respective microbiome compositions. RESULTS: Dysbiosis in Winnie mice was already established at 4 weeks of age, before histologic evidence of gut inflammatory changes, in which microbial communities diverged from that derived from their mothers. Furthermore, dysbiosis persisted until 12 weeks of age, with peak differences in microbiome composition observed between Winnie and WT mice at 8 weeks of age. The relative abundance of Bacteroidetes was greater in Winnie compared with WT mice. Verrucomicrobia was detected at the highest relative levels in 4-week-old Winnie mice; in particular, Akkermansia muciniphila was among the most abundant species found at 4 weeks of age. CONCLUSIONS: Our results demonstrate that mutant genetic determinants involved in the complex regulation of intestinal homeostasis, such as that observed in Winnie mice, are able to promote early gut dysbiosis that is independent from maternal microbial transfer, including breastfeeding. Our data provide evidence for intestinal dysbiosis attributed to a Muc2-driven mucus defect that leads to colonic inflammation and may represent an important target for the design of future interventional studies.

The Epstein-Barr virus encoded LMP1 oncoprotein modulates cell adhesion via regulation of activin A/TGFß and ß1 integrin signalling.

Approximately 20% of global cancer incidence is causally linked to an infectious agent. Epstein-Barr virus (EBV) accounts for around 1% of all virus-associated cancers and is associated with nasopharyngeal carcinoma (NPC). Latent membrane protein 1 (LMP1), the major oncoprotein encoded by EBV, behaves as a constitutively active tumour necrosis factor (TNF) receptor activating a variety of signalling pathways, including the three classic MAPKs (ERK-MAPK, p38 MAPK and JNK/SAPK). The present study identifies novel signalling properties for this integral membrane protein via the induction and secretion of activin A and TGFß1, which are both required for LMP1's ability to induce the expression of the extracellular matrix protein, fibronectin. However, it is evident that LMP1 is unable to activate the classic Smad-dependent TGFß signalling pathway, but rather elicits its effects through the non-Smad arm of TGFß signalling. In addition, there is a requirement for JNK/SAPK signalling in LMP1-mediated fibronectin induction. LMP1 also induces the expression and activation of the major fibronectin receptor, α5ß1 integrin, an effect that is accompanied by increased focal adhesion formation and turnover. Taken together, these findings support the putative role for LMP1 in the pathogenesis of NPC by contributing to the metastatic potential of epithelial cells.