IL-6 Signaling Regulates Small Intestinal Crypt Homeostasis.

Gut homeostasis is a tightly regulated process requiring finely tuned complex interactions between different cell types, growth factors, or cytokines and their receptors. Previous work has implicated a role for IL-6 and mucosal immune cells in intestinal regeneration following injury and in promoting inflammation and cancer. We hypothesized that IL-6 signaling could also modulate crypt homeostasis. Using mouse in vitro crypt organoid and in vivo models, this study first demonstrated that exogenous IL-6 promoted crypt organoid proliferation and increased stem cell numbers through pSTAT3 activation in Paneth cells. Immunolabeling studies showed that the IL-6 receptor was restricted to the basal membrane of Paneth cells both in vitro and in vivo and that the crypt epithelium also expressed IL-6. Either a blocking Ab to the IL-6 receptor or a neutralizing Ab to IL-6 significantly reduced in vitro basal crypt organoid proliferation and budding, and in vivo significantly reduced the number of nuclei and the number of Lgr5EGFP-positive stem cells per crypt compared with IgG-treated mice, with the number of Paneth cells per crypt also significantly reduced. Functional studies demonstrated that IL-6-induced in vitro crypt organoid proliferation and crypt budding was abrogated by the Wnt inhibitor IWP2. This work demonstrates that autocrine IL-6 signaling in the gut epithelium regulates crypt homeostasis through the Paneth cells and the Wnt signaling pathway.

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.

Luminal microbes promote monocyte-stem cell interactions across a healthy colonic epithelium.

The intestinal epithelium forms a vital barrier between luminal microbes and the underlying mucosal immune system. Epithelial barrier function is maintained by continuous renewal of the epithelium and is pivotal for gut homeostasis. Breaching of the barrier causes mobilization of immune cells to promote epithelial restitution. However, it is not known whether microbes at the luminal surface of a healthy epithelial barrier influence immune cell mobilization to modulate tissue homeostasis. Using a mouse colonic mucosal explant model, we demonstrate that close proximity of luminal microbes to a healthy, intact epithelium results in rapid mucus secretion and movement of Ly6C(+)7/4(+) monocytes closer to epithelial stem cells. These early events are driven by the epithelial MyD88-signaling pathway and result in increased crypt cell proliferation and intestinal stem cell number. Over time, stem cell number and monocyte-crypt stem cell juxtapositioning return to homeostatic levels observed in vivo. We also demonstrate that reduced numbers of tissue Ly6C+ monocytes can suppress Lgr5EGFP+ stem cell expression in vivo and abrogate the response to luminal microbes ex vivo. The functional link between monocyte recruitment and increased crypt cell proliferation was further confirmed using a crypt-monocyte coculture model. This work demonstrates that the healthy gut epithelium mediates communication between luminal bacteria and monocytes, and monocytes can modulate crypt stem cell number and promote crypt cell proliferation to help maintain gut homeostasis.

Canonical Wnt signals combined with suppressed TGFß/BMP pathways promote renewal of the native human colonic epithelium.

BACKGROUND: A defining characteristic of the human intestinal epithelium is that it is the most rapidly renewing tissue in the body. However, the processes underlying tissue renewal and the mechanisms that govern their coordination have proved difficult to study in the human gut. OBJECTIVE: To investigate the regulation of stem cell-driven tissue renewal by canonical Wnt and TGFß/bone morphogenetic protein (BMP) pathways in the native human colonic epithelium. DESIGN: Intact human colonic crypts were isolated from mucosal tissue samples and placed into 3D culture conditions optimised for steady-state tissue renewal. High affinity mRNA in situ hybridisation and immunohistochemistry were complemented by functional genomic and bioimaging techniques. The effects of signalling pathway modulators on the status of intestinal stem cell biology, crypt cell proliferation, migration, differentiation and shedding were determined. RESULTS: Native human colonic crypts exhibited distinct activation profiles for canonical Wnt, TGFß and BMP pathways. A population of intestinal LGR5/OLFM4-positive stem/progenitor cells were interspersed between goblet-like cells within the crypt-base. Exogenous and crypt cell-autonomous canonical Wnt signals supported homeostatic intestinal stem/progenitor cell proliferation and were antagonised by TGFß or BMP pathway activation. Reduced Wnt stimulation impeded crypt cell proliferation, but crypt cell migration and shedding from the crypt surface were unaffected and resulted in diminished crypts. CONCLUSIONS: Steady-state tissue renewal in the native human colonic epithelium is dependent on canonical Wnt signals combined with suppressed TGFß/BMP pathways. Stem/progenitor cell proliferation is uncoupled from crypt cell migration and shedding, and is required to constantly replenish the crypt cell population.

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.

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.

Hypoxia-induced neutrophil survival is mediated by HIF-1alpha-dependent NF-kappaB activity.

Neutrophils are key effector cells of the innate immune response and are required to migrate and function within adverse microenvironmental conditions. These inflammatory sites are characterized by low levels of oxygen and glucose and high levels of reductive metabolites. A major regulator of neutrophil functional longevity is the ability of these cells to undergo apoptosis. We examined the mechanism by which hypoxia causes an inhibition of neutrophil apoptosis in human and murine neutrophils. We show that neutrophils possess the hypoxia-inducible factor (HIF)-1alpha and factor inhibiting HIF (FIH) hydroxylase oxygen-sensing pathway and using HIF-1alpha-deficient myeloid cells demonstrate that HIF-1alpha is directly involved in regulating neutrophil survival in hypoxia. Gene array, TaqMan PCR, Western blotting, and oligonucleotide binding assays identify NF-kappaB as a novel hypoxia-regulated and HIF-dependent target, with inhibition of NF-kappaB by gliotoxin or parthenolide resulting in the abrogation of hypoxic survival. In addition, we identify macrophage inflammatory protein-1beta as a novel hypoxia-induced neutrophil survival factor.

Failure of bone morphogenetic protein receptor trafficking in pulmonary arterial hypertension: potential for rescue.

Heterozygous germline mutations in the gene encoding the bone morphogenetic protein type II receptor cause familial pulmonary arterial hypertension (PAH). We previously demonstrated that the substitution of cysteine residues in the ligand-binding domain of this receptor prevents receptor trafficking to the cell membrane. Here we demonstrate the potential for chemical chaperones to rescue cell-surface expression of mutant BMPR-II and restore function. HeLa cells were transiently transfected with BMPR-II wild type or mutant (C118W) receptor constructs. Immunolocalization studies confirmed the retention of the cysteine mutant receptor mainly in the endoplasmic reticulum. Co-immunoprecipitation studies of Myc-tagged BMPR-II confirmed that the cysteine-substituted ligand-binding domain mutation, C118W, is able to associate with BMP type I receptors. Furthermore, following treatment with a panel of chemical chaperones (thapsigargin, glycerol or sodium 4-phenylbutyrate), we demonstrated a marked increase in cell-surface expression of mutant C118W BMPR-II by FACS analysis and confocal microscopy. These agents also enhanced the trafficking of wild-type BMPR-II, though to a lesser extent. Increased cell-surface expression of mutant C118W BMPR-II was associated with enhanced Smad1/5 phosphorylation in response to BMPs. These findings demonstrate the potential for rescue of mutant BMPR-II function from the endoplasmic reticulum. For the C118W mutation in the ligand-binding domain of BMPR-II, cell-surface rescue leads to at least partial restoration of BMP signalling. We conclude that enhancement of cell-surface trafficking of mutant and wild-type BMPR-II may have therapeutic potential in familial PAH.

Mutations in bone morphogenetic protein type II receptor cause dysregulation of Id gene expression in pulmonary artery smooth muscle cells: implications for familial pulmonary arterial hypertension.

Heterozygous germ line mutations in the gene encoding the bone morphogenetic protein (BMP) type II receptor occur in more than 80% of patients with familial pulmonary arterial hypertension. Because inhibitors of DNA binding (Id) genes are major targets of BMP/Smad signaling, we studied the regulation of these transcription factors in pulmonary artery smooth muscle cells harboring mutations in BMP type II receptor and control cells. Mutant cells demonstrated a marked deficiency in BMP4-stimulated Id1 and Id2 gene and protein expression compared with control cells. Mutant cells were deficient in Smad1/5 signaling in response to BMPs but also in extracellular signal-regulated kinase (ERK)1/2 activation. We provide evidence for an important interaction between Smad1/5 and ERK1/2 signaling in the regulation of Id gene expression. Thus, BMP4-induced Id1 expression was negatively regulated by ERK1/2 activation. The mechanism involves ERK1/2-dependent phosphorylation of the Smad1 linker region (serine 206), which limits C-terminal serine 463/465 phosphorylation and inhibits Smad nuclear accumulation. Furthermore, activation of ERK1/2 by platelet-derived growth factor BB also caused Smad1 linker region phosphorylation and inhibited BMP4-induced Id1 gene expression. In contrast, Id2 expression was positively regulated by ERK1/2. Moreover, we show that both BMP type II receptor mutation and Id1 knockdown leads to loss of growth suppression by BMPs. Taken together, these findings indicate an important interaction between ERK1/2 and Smad1/5 in the regulation of Id genes. Platelet-derived growth factor, via ERK1/2, further impairs the deficiency in Smad signaling found in BMP type II receptor mutant cells. The integration of these signals at the level of Id gene expression may contribute to the pathogenesis of familial pulmonary arterial hypertension.

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.

A Bronze-Tomato Enriched Diet Affects the Intestinal Microbiome under Homeostatic and Inflammatory Conditions.

Inflammatory bowel diseases (IBD) are debilitating chronic inflammatory disorders that develop as a result of a defective immune response toward intestinal bacteria. Intestinal dysbiosis is associated with the onset of IBD and has been reported to persist even in patients in deep remission. We investigated the possibility of a dietary-induced switch to the gut microbiota composition using Winnie mice as a model of spontaneous ulcerative colitis and chow enriched with 1% Bronze tomato. We used the near isogenic tomato line strategy to investigate the effects of a diet enriched in polyphenols administered to mild but established chronic intestinal inflammation. The Bronze-enriched chow administered for two weeks was not able to produce any macroscopic effect on the IBD symptoms, although, at molecular level there was a significant induction of anti-inflammatory genes and intracellular staining of T cells revealed a mild decrease in IL17A and IFNγ production. Analysis of the microbial composition revealed that two weeks of Bronze enriched diet was sufficient to perturb the microbial composition of Winnie and control mice, suggesting that polyphenol-enriched diets may create unfavorable conditions for distinct bacterial species. In conclusion, dietary regimes enriched in polyphenols may efficiently support IBD remission affecting the intestinal dysbiosis.

Secretory Leukoprotease Inhibitor (Slpi) Expression Is Required for Educating Murine Dendritic Cells Inflammatory Response Following Quercetin Exposure.

Dendritic cells' (DCs) ability to present antigens and initiate the adaptive immune response confers them a pivotal role in immunological defense against hostile infection and, at the same time, immunological tolerance towards harmless components of the microbiota. Food products can modulate the inflammatory status of intestinal DCs. Among nutritionally-derived products, we investigated the ability of quercetin to suppress inflammatory cytokines secretion, antigen presentation, and DCs migration towards the draining lymph nodes. We recently identified the Slpi expression as a crucial checkpoint required for the quercetin-induced inflammatory suppression. Here we demonstrate that Slpi-KO DCs secrete a unique panel of cytokines and chemokines following quercetin exposure. In vivo, quercetin-enriched food is able to induce Slpi expression in the ileum, while little effects are detectable in the duodenum. Furthermore, Slpi expressing cells are more frequent at the tip compared to the base of the intestinal villi, suggesting that quercetin exposure could be more efficient for DCs projecting periscopes in the intestinal lumen. These data suggest that quercetin-enriched nutritional regimes may be efficient for suppressing inflammatory syndromes affecting the ileo-colonic tract.

Eotaxin-1/CC chemokine ligand 11: a novel eosinophil survival factor secreted by human pulmonary artery endothelial cells.

Airway eosinophilia plays a major role in the pathogenesis of asthma with the inhibition of apoptosis by GM-CSF and IL-5 proposed as a mechanism underlying prolonged eosinophil survival. In vivo and ex vivo studies have indicated the capacity of interventions that drive human eosinophil apoptosis to promote the resolution of inflammation. Far less is known about the impact of transendothelial migration on eosinophil survival, in particular, the capacity of endothelial cell-derived factors to contribute toward the apoptosis-resistant phenotype characteristic of airway-resident eosinophils. We examined the effects of conditioned medium from human pulmonary artery endothelial cells (HPAEC-CM) on eosinophil apoptosis in vitro. HPAEC-CM inhibited eosinophil, but not neutrophil apoptosis. This effect was specific to HPAECs and comparable in efficacy to the survival effects of GM-CSF and IL-5. The HPAEC survival factor was shown, on the basis of GM-CSF, IL-5, and IL-3 detection assays, Ab neutralization, and sensitivity to PI3K inhibition, to be clearly discrete from these factors. Gel filtration of HPAEC-CM revealed a peak of eosinophil survival activity at 8-12 kDa, and PCR confirmed the presence of mRNA for CCL5, CCL11, CCL24, CCL26, and CCL27 in the HPAECs. The CCR3 antagonist GW782415 caused a major inhibition of the HPAEC-CM-induced survival effect, and Ab neutralization of individual CCR3 chemokines revealed CCL11 as the major survival factor present in the HPAEC-CM. Furthermore, chemokine Ab arrays demonstrated up-regulation of CCL11 in HPAEC-CM. These data demonstrate the capacity of HPAECs to generate CCR3 agonists and the ability of CCL11 to inhibit human eosinophil apoptosis.

Aminopeptidase N (CD13) regulates tumor necrosis factor-alpha-induced apoptosis in human neutrophils.

Neutrophil apoptosis plays a central role in the resolution of granulocytic inflammation. We have shown previously that tumor necrosis factor-alpha (TNFalpha) enhances the rate of neutrophil apoptosis at early time points via a mechanism involving both TNF receptor (TNFR) I and TNFRII. Here we reveal a marked but consistent variation in the magnitude of the pro-apoptotic effect of TNFalpha in neutrophils isolated from healthy donors, and we show that inhibition of cell surface aminopeptidase N (APN) using actinonin, bestatin, or inhibitory peptides significantly enhanced the efficacy of TNFalpha-induced killing. Notably, an inverse correlation is shown to exist between neutrophil APN activity and the sensitivity of donor cells to TNFalpha-induced apoptosis. Inhibition of cell surface APN appears to interfere with the shedding of TNFRI, and as a consequence results in augmented TNFalpha-induced apoptosis, cell polarization, and TNFalpha-primed, formyl-methionyl-leucyl-phenylalanine-stimulated respiratory burst. Of note, actinonin and bestatin had no effect on TNFRII expression under resting or TNFalpha-stimulated conditions and did not alter CXCRI or CXCRII expression. These data suggest significant variation in the activity of APN/CD13 on the cell surface of neutrophils in normal individuals and reveal a novel mechanism whereby APN/CD13 regulates TNFalpha-induced apoptosis via inhibition of TNFRI shedding. This has therapeutic relevance for driving neutrophil apoptosis in vivo.

Mechanism of cicaprost-induced desensitization in rat pulmonary artery smooth muscle cells involves a PKA-mediated inhibition of adenylyl cyclase.

Long-term infusion of prostacyclin, or its analogs, is an effective treatment for severe pulmonary arterial hypertension. However, dose escalation is often required to maintain efficacy. The aim of this study was to investigate the mechanisms of prostacyclin receptor desensitization using the prostacyclin analog cicaprost in rat pulmonary artery smooth muscle cells (PASMCs). Desensitization of the cAMP response occurred in 63 nM cicaprost after a 6-h preincubation with agonist. This desensitization was reversed 12 h after agonist removal, and resensitization was inhibited by 10 microg/ml of cycloheximide. Desensitization was heterologous since desensitization to other G(s)alpha-adenylyl cyclase (AC)-coupled agonists, isoproterenol (1 microM), adrenomedullin (100 nM), or bradykinin (1 microM), was also reduced by preincubation with cicaprost. The reduced cAMP response to prolonged cicaprost exposure appeared to be due to inhibition of AC activity since the responses to the directly acting AC agonist forskolin (3 microM) and the selective AC5 activator NKH-477 were similarly reduced. Expression of AC2 and AC5/6 protein levels transiently decreased after 1 h of cicaprost exposure. The PKA inhibitor H-89 (1 microM) added 1 h before cicaprost preincubation (6 h, 63 nM) completely reversed cicaprost-induced desensitization, whereas the PKC inhibitor bisindolylmaleimide (100 nM) was only partly effective. Desensitization was not prevented by the G(i) inhibitor pertussis toxin. In conclusion, chronic treatment of PASMCs with cicaprost induced heterologous, reversible desensitization by inhibition of AC activity. Our data suggest that heterologous G(s)alpha desensitization by cicaprost is mediated predominantly by a PKA-inhibitable isoform of AC, most likely AC5/6.