Intestinal Organoids as a Tool for Inflammatory Bowel Disease Research.

Inflammatory Bowel Diseases (IBD) are difficult to model as freshly acquired tissues are short-lived, provide data as a snapshot in time, and are not always accessible. Many patients with IBD are non-responders to first-line treatments, and responders are prone to developing resistance to treatment over time-resulting in reduced patient quality of life, increased time to remission, and potential relapse. IBD is heterogenous and we are yet to fully understand the mechanisms of disease; thus, our ability to diagnose and prescribe optimal treatment remains ineffective. Intestinal organoids are derived from patient tissues expanded in vitro. Organoids offer unique insight into individual patient disease and are a potential route to personalized treatments. However, organoid models do not contain functional microbial and immune cell components. In this review, we discuss immune cell subsets in the context of IBD, and the requirement of immune cell and microbial components in organoid models for IBD research.

cAMP-dependent secretagogues stimulate the NaHCO3 cotransporter in the villous epithelium of the brushtail possum, Trichosurus vulpecula.

In the ileum of the brushtail possum, Trichosurus vulpecula, fluid secretion appears to be driven by electrogenic HCO3- secretion. Consistent with this, the cystic fibrosis transmembrane conductance regulator is expressed in the apical membrane of the ileal epithelial cells and the pancreatic or secretory variant of the NaHCO3 cotransporter in the basolateral membrane. This suggests that in the possum ileum, electrogenic HCO3- secretion is driven by basolateral NaHCO3 cotransporter (NBC) activity. To determine if the NBC contributes to HCO3- secretion in the possum ileum, intracellular pH (pHi) measurements in isolated villi were used to demonstrate NBC activity in the ileal epithelial cells and investigate the effect of cAMP-dependent secretagogues. In CO2/HCO3--free solutions, recovery of the epithelial cells from an acid load was Na+-dependent and ≈80% inhibited by ethyl-isopropyl-amiloride (EIPA, 10 µmol L-1), indicative of the presence of an Na+/H+ exchanger, most likely NHE1. However, in the presence of CO2/HCO3-, EIPA only inhibited ≈ 50% of the recovery, the remainder was inhibited by 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS, 500 µmol L-1), indicative of NBC activity. Under steady-state conditions, NHE1 inhibition by EIPA had little effect on pHi in the presence or absence of secretagogues, but NBC inhibition with DIDS resulted in a rapid acidification of the cells, which was increased fivefold by secretagogues. These data demonstrate the functional activity of an NaHCO3 cotransporter in the ileal epithelial cells. Furthermore, the stimulation of NBC activity by secretagogues is consistent with the involvement of an NaHCO3 cotransporter in electrogenic HCO3- secretion.

Distinct immune signatures in the colon of Crohn's disease and ankylosing spondylitis patients in the absence of inflammation.

Crohn's disease (CD) is an inflammatory bowel disease characterized by patchy inflammation of the gastrointestinal tract. Ankylosing spondylitis (AS) is primarily characterized by inflammation of the lower vertebral column, and many patients with AS present with inflammatory gut symptoms. Genome-wide association studies have highlighted significant overlap in short nucleotide polymorphisms for both diseases. We hypothesized that patients with CD and AS have a common intestinal immune signature, characterized by inflammatory T cells, compared with healthy people. We designed a pilot study to determine both the feasibility of defining complex immune signatures from primary tissue, and differences in the local immune signature of people with inflammatory diseases compared with healthy people. Intestinal biopsies were obtained by colonoscopy from healthy patients, non-inflamed regions of CD patients and AS patients with inflammatory gut symptoms. A flow cytometry platform was developed measuring polyfunctional T-cell populations based on cytokines, surface molecules and transcription factors. There was overlap in the immune signature of people with CD or AS, characterized by changes in the frequency of regulatory T cells, compared with healthy people. There were significant differences in frequencies of other polyfunctional T-cell populations-CD patients had an increased frequency of T cells producing interleukin-22 (IL-22) and interferon-γ, whereas AS patients had an increased frequency of T cells producing IL-2; compared with healthy people. These data indicate that the local immune signature could be described in these patients and that distinct immune mechanisms may underlie disease progression.

Aggravation of Established Colitis in Specific Pathogen-free IL-10 Knockout Mice by Restraint Stress Is Not Mediated by Increased Colonic Permeability.

BACKGROUND: Pathogenic mechanisms responsible for the undulating symptom pattern, or indeed causative agents for the development, of inflammatory bowel diseases [IBD] are largely unknown. Many physicians and most patients are convinced that stress affects the course of IBD. As with most factors that contribute to IBD, it is unclear whether stress merely exacerbates established disease or indeed contributes to the development of disease. We designed this study to investigate whether stress induces or aggravates colitis in interkeukin-10 knockout [IL-10 ko] mice and to determine the role of intestinal permeability in this model of stress-related colitis. METHODS: The study was divided into two experiments depending on the age of the animals. Stress was induced by placing 5-week old disease-free mice or 8-week-old mice (IL-10ko and wild type [wt]) with mild colitis in movement restrainers for 2h twice daily for 7 days. The development of colitis was assessed clinically [weight and faecal pellet production], histologically [haematoxylin and eosin staining], and biochemically [colonic IL-2, IL-4, IL-6, IL-12p40, TNFα, and IFNγ]. Permeability was measured in Ussing chambers. RESULTS: Faecal pellet production increased significantly in all stressed animals compared with control animals, indicating successful application of stress. Stressed 8-week old mice lost weight [p < 0.001] and stressed IL-10(-/-) mice showed a significantly increased histological score compared with non-stressed or wt mice [p < 0.001]. There was no appreciable difference in cytokine production. Stress did not alter intestinal permeability. CONCLUSIONS: Restraint stress aggravates experimental colitis in 8-week old IL-10ko mice but cannot induce colitis in disease-free younger mice. This is not mediated by an increased intestinal permeability.

Glucose transport into everted sacs of the small intestine of mice.

The Na(+)-glucose cotransporter is a key transport protein that is responsible for absorbing Na(+) and glucose from the luminal contents of the small intestine and reabsorption by the proximal straight tubule of the nephron. Robert K. Crane originally described the cellular model of absorption of Na(+) and glucose by a "cotransport process" in 1960. Over the past 50+ yr, numerous groups have tested and verified Crane's hypothesis. Eventually, Wright and colleagues cloned the Na(+)-glucose cotransporter (SGLT1; the product of the SLC5A1 gene) in 1987. This article provides a "hands-on" laboratory exercise using the everted mouse jejunal preparation (everted sac) that allows students to investigate various components of the Na(+)-glucose cotransport absorptive cell model (e.g., Na(+) dependence of SGLT1, inhibition of SGLT1, and inhibition of Na(+)-K(+)-ATPase). Additionally, the laboratory exercise includes a case-based study of glucose-galactose malabsorption in which the students conduct an internet search and participate in a small-group discussion during the laboratory period to better understand the basic principles and functions of the Na(+)-glucose absorptive process of the small intestine. This laboratory exercise was introduced into the second-year undergraduate physiology curriculum in 2008, and >850 physiology students have participated in this laboratory exercise. The students have produced very robust and reproducible data that clearly illustrate the theory of the cellular model for Na(+)-glucose absorption by the jejunum.

Is the north to south gradient in inflammatory bowel disease a global phenomenon?

Inflammatory bowel diseases (IBDs), such as Crohn's disease and ulcerative colitis, are multifactorial diseases that result from an overly aggressive immune response towards an environmental trigger in a genetically susceptible host. Much has been learned about susceptibility genes; however, environmental triggers are still largely unknown. In the 1990s, a large study from Europe suggested a north-south gradient regarding the incidence of IBD; however, this was never shown convincingly for the USA. In the present study, the authors used and extended data from the Nurses' Health Study I and II and were able to demonstrate that indeed the risk of developing IBD is significantly lower for the southern latitudes of the USA and did not depend on ancestry or lifestyle factors such as smoking. The authors speculate that an increased exposure to sunlight provides protection from IBD via the vitamin D pathway.

CFTR is restricted to a small population of high expresser cells that provide a forskolin-sensitive transepithelial Cl- conductance in the proximal colon of the possum, Trichosurus vulpecula.

The cystic fibrosis transmembrane conductance regulator (CFTR) is central to anion secretion in both the possum and eutherian small intestine. Here, we investigated its role in the possum proximal colon, which has novel transport properties compared with the eutherian proximal colon. Despite considerable CFTR expression, high doses of the CFTR activator forskolin (EC(50)≈10 µmol l(-1)) were required for a modest, CFTR-dependent increase in short-circuit current (I(sc)) in the proximal colon. Presumably, this is because CFTR is restricted to the apical membrane of a small population of CFTR high expresser (CHE) cells in the surface and upper crypt epithelium. Furthermore, although the forskolin-stimulated I(sc) was dependent on serosal Na(+), Cl(-) and HCO(3)(-), consistent with anion secretion, inhibition of the basolateral Na-K-2Cl(-) (NKCC1) or Na-HCO(3) (pNBCe1) cotransporters did not prevent it. Therefore, although NKCC1 and pNBCe1 are expressed in the colonic epithelium they do not appear to be expressed in CHE cells. At low doses (IC(50)≈1 µmol l(-1)), forskolin also decreased the transepithelial conductance (G(T)) of the colon through inhibition of a 4,4'-diisothiocyano-2,2'-stilbenedisulphonic acid-sensitive anion conductance in the basolateral membrane of the CHE cells. This conductance is arranged in series with CFTR in the CHE cells and, therefore, the CHE cells provide a transepithelial Cl(-) conductance for passive Cl(-) absorption across the epithelium. Inhibition of the basolateral Cl(-) conductance of the CHE cells by forskolin will inhibit Na(+) absorption by restricting the movement of its counter-ion Cl(-), assisting in the conversion of the tissue from an absorptive to a secretory state.

COMMD1 downregulates the epithelial sodium channel through Nedd4-2.

The epithelial sodium channel (ENaC) is important for the long-term control of Na(+) homeostasis and blood pressure. Our previous studies demonstrated that Copper Metabolism Murr1 Domain-containing protein 1 (COMMD1; previously known as Murr1), a protein involved in copper metabolism, inhibited amiloride-sensitive current in Xenopus laevis oocytes expressing ENaC (J Biol Chem 279: 5429, 2004). In this study, we report that COMMD1 inhibits amiloride-sensitive current in mammalian epithelial cells expressing ENaC, that the COMM domain of COMMD1 is sufficient for this effect, and that knockdown of COMMD1 increases amiloride-sensitive current. COMMD1 is coexpressed with ENaC in rat kidney medulla cells. COMMD1 increased ubiquitin modification of ENaC and decreased its cell surface expression. COMMD1 abolished insulin-stimulated amiloride-sensitive current and attenuated the stimulation of current by activated serum and glucocorticoid-regulated kinase (SGK1). COMMD1 was found to interact with both SGK1 and Akt1/protein kinase B, and knockdown of COMMD1 enhanced the stimulatory effect of both SGK1 and Akt1 on amiloride-sensitive current. COMMD1's effects were reduced in the presence of ENaC proteins containing PY motif mutations, abolished in the presence of a dominant negative form of Nedd4-2, and knockdown of COMMD1 reduced the inhibitory effect of Nedd4-2 on ENaC, but did not enhance current when Nedd4-2 was knocked down. These data suggest that COMMD1 modulates Na(+) transport in epithelial cells through regulation of ENaC cell surface expression and this effect is likely mediated via Nedd4-2.

Secretagogues stimulate electrogenic HCO3- secretion in the ileum of the brushtail possum, Trichosurus vulpecula: evidence for the role of a Na+/HCO3- cotransporter.

Fluid secretion is essential for intestinal function and, in eutherian mammals, is driven by electrogenic Cl(-) transport, which is dependent upon a bumetanide-sensitive, basolateral Na(+)/K(+)/2 Cl(-) cotransporter, NKCC1. However, ileal secretion in the brushtail possum, a marsupial, involves a fundamentally different process, since NKCC1 expression is low in this tissue and the secretagogue-induced short circuit current (I(sc)) is insensitive to bumetanide. In view of these differences we have investigated the basis of the secretory response of the possum ileum. In the Ussing chamber the secretory I(sc) is independent of Cl(-) but dependent upon Na(+) and serosal HCO(3)(-)/CO(2), suggesting that secretagogues stimulate electrogenic HCO(3)(-) secretion. In agreement with this, serosal DIDS (4,4'-diisothiocyano-stilbene-2,2'-disulfonate; 1 mmol l(-1)) inhibited the secretory response. However, acetazolamide (1 mmol l(-1)) and serosal amiloride (1 mmol l(-1)) had little effect, indicating that HCO(3)(-) secretion is driven by HCO(3)(-) transport from the serosal solution into the cell, rather than hydration of CO(2) by carbonic anhydrase. Consistent with this the pancreatic variant of the electrogenic Na(+)/HCO(3)(-) cotransporter (pNBC) is highly expressed in the ileal epithelium and is located in the basolateral membrane of the epithelial cells, predominantly in the mid region of the villi, with lower levels of expression in the crypts and no expression in the villous tips. We conclude that the secretory response of the possum ileum involves electrogenic HCO(3)(-) secretion driven by a basolateral pNBC and that the ileal HCO(3)(-) secretion is associated with a specialised function of the possum ileum, most probably related to hindgut fermentation.

Electrogenic Cl(-) secretion does not occur in the ileum of the Australian common brushtail possum, Trichosurus vulpecula, due to low levels of expression of the NaK2Cl cotransporter, NKCC1.

The colon of the brushtail possum does not have an electrogenic secretory response. Given the functional significance of electrogenic Cl(-) secretion in the intestine of eutherian mammals, we have investigated the secretory response in the small intestine of this marsupial. In the Ussing chamber cAMP-dependent secretagogues stimulated a sustained increase in ileal short-circuit current (Isc), whereas Ca(2+)-dependent secretagogues induced a transient increase. Both the responses were inhibited by mucosal addition of the anion channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid (100 mciromol l(-1)), consistent with an anion secretory response. However, the responses were not inhibited by serosal bumetanide (10 mciromol l(-1)) and were independent of bath Cl(-), indicating that the stimulated ileal Isc does not involve electrogenic Cl(-) secretion driven by the NaK2Cl cotransporter, NKCC1. Consistent with this, there were low levels of NKCC1 expression in the ileal epithelium. In particular, NKCC1 expression in the ileal crypt cells was comparable to that of the villous cells. This differs from eutherian mammals where high levels of NKCC1 expression in the ileal crypt cells are associated with their role in Cl(-) secretion. The cAMP- and Ca(2+)-dependent secretory responses were inhibited by the removal of HCO(3) (-) suggesting that these responses were due to electrogenic HCO(3) (-) secretion. We conclude that the ileum of the possum does not secrete Cl(-) due to low levels of NKCC1 expression. It does however appear to secrete HCO(3) (-). These results are further significant examples of differences in the transport function of the possum intestinal epithelium compared with eutherian mammals.

Methoxsalen stimulates electrogenic Cl- secretion in the mouse jejunum.

We used the short-circuit current (I(sc)) and patch-clamp techniques to investigate the effects of methoxsalen (MTX) on the electrogenic Cl- secretion of the mouse jejunum. MTX stimulated a sustained increase in Isc that was dose dependent. Bumetanide inhibited MTX-stimulated Isc in a dose-dependent manner consistent with activation of Cl- secretion. MTX failed to stimulate I(sc) following maximal activation of the cAMP pathway by forskolin, but did increase Isc after a submaximal dose of forskolin. Glibenclamide, a blocker of the cystic fibrosis transmembrane conductance regulator (CFTR), reduced the MTX-stimulated increase of Isc by 59 +/- 6%. The cAMP-dependent K+ channel blocker 293B did not alter the MTX-activated I(sc); however, clotrimazole, an intermediate Ca2(+)-activated K+ channel (IK(Ca)) blocker, reduced the MTX-stimulated I(sc). MTX did not alter Na(+)-glucose cotransport across the mouse jejunum. In cell-attached membrane patches, MTX increased the open probability of the basolateral IK(Ca) channel of isolated crypts. These data suggest that the CFTR and IK(Ca) channels participate in the MTX-activated, sustained Cl- secretory response of the mouse jejunum.