Mast Cell Mediated Regulation of Small Intestinal Chloride Malabsorption in SAMP1/YitFc Mouse Model of Spontaneous Chronic Ileitis

In Inflammatory Bowel Disease (IBD), malabsorption of electrolytes (NaCl) results in diarrhea. Inhibition of coupled NaCl absorption, mediated by the dual operation of Na:H and Cl:HCO3 exchangers on the brush border membrane (BBM) of the intestinal villus cells has been reported in IBD. In the SAMP1/YitFcs (SAMP1) mice model of spontaneous ileitis, representing Crohn’s disease, DRA (Downregulated in Adenoma) mediated Cl:HCO3 exchange was shown to be inhibited secondary to diminished affinity of the exchanger for Cl. However, NHE3 mediated Na:H exchange remained unaffected. Mast cells and their secreted mediators are known to be increased in the IBD mucosa and can affect intestinal electrolyte absorption. However, how mast cell mediators may regulate Cl:HCO3 exchange in SAMP1 mice is unknown. Therefore, the aim of this study was to determine the effect of mast cell mediators on the downregulation of DRA in SAMP1 mice. Mast cell numbers and their degranulation marker enzyme (β-hexosaminidase) levels were significantly increased in SAMP1 mice compared to control AKR mice. However, treatment of SAMP1 mice with a mast cell stabilizer, ketotifen, restored the β-hexosaminidase enzyme levels to normal in the intestine, demonstrating stabilization of mast cells by ketotifen. Moreover, downregulation of Cl:HCO3 exchange activity was restored in ketotifen treated SAMP1 mice. Kinetic studies showed that ketotifen restored the altered affinity of Cl:HCO3 exchange in SAMP1 mice villus cells thus reinstating its activity to normal. Further, RT-qPCR, Western blot and immunofluorescence studies showed that the expression levels of DRA mRNA and BBM protein, respectively remained unaltered in all experimental conditions, supporting the kinetic data. Thus, inhibition of Cl:HCO3 exchange resulting in chloride malabsorption leading to diarrhea in IBD is likely mediated by mast cell mediators.

Depletion of Intestinal Stem Cell Niche Factors Contributes to the Alteration of Epithelial Differentiation in SAMP1/YitFcsJ Mice With Crohn Disease-Like Ileitis

Background SAMP1/YitFcsJ (SAMP1) mice spontaneously develop terminal ileitis resembling human Crohn disease. SAMP1 mice have exhibited alteration of epithelial cell lineage distribution and an overall proliferation of the crypt cell population; however, it has not been evaluated whether epithelial differentiation is impaired because of dysfunction of intestinal stem cells (ISCs) or their niche factors. Methods Using the intestine of SAMP1 mice aged 10 to 14 weeks, morphometric alterations in the crypt-villus architecture, ISCs, crypt cells, and differentiated cells; organoid formation capacity of intestinal crypts; and niche signaling pathways were analyzed and compared with those of age-matched control AKR/J (AKR) mice. Results The ileum of SAMP1 mice showed increased depth of intestinal crypts and decreased surface area of the villi compared with those in the ileum of AKR mice. The number of ISCs in the ileal crypts did not differ between SAMP1 and AKR mice; however, the number of Paneth cells decreased and the number of transient amplifying cells increased. The organoid formation rate of the ileal crypts of SAMP1 mice decreased significantly compared with that of AKR mice. The performance of RNA sequencing for intestinal crypts found that the expression of ISC niche factors, such as Wnt3, Dll1, and Dll4, was decreased significantly in the ileal crypts of SAMP1 mice compared with those of AKR mice. Among the ISC niche signals, the Notch signaling-related genes tended to be downregulated. In particular, immunocytochemistry revealed that the expression of Paneth cell–expressing Notch ligand Dll4 was significantly decreased in the intestinal tissue and organoids of SAMP1 mice compared with those of AKR mice. Conclusions Depletion of niche factors for ISCs contributes to the alteration of epithelial differentiation in SAMP1 mice.

Inducible Nitric Oxide Regulates Na-Glucose Co-Transport in a Spontaneous SAMP1/YitFc Mouse Model of Chronic Ileitis

In mammalian small intestine, glucose is primarily absorbed via Na-dependent glucose co-transporter (SGLT1) on the brush border membrane (BBM) of absorptive villus cells. Malabsorption of nutrients (e.g., glucose) leads to malnutrition, a common symptom of inflammatory bowel disease (IBD), where the mucosa is characterized by chronic inflammation. Inducible nitric oxide (iNO) is known to be elevated in IBD mucosa. SAMP1/YitFc (SAMP1) mouse is a spontaneous model of chronic ileitis that develops lesions in its terminal ileum, very similar to human IBD. How SGLT1 may be affected in SAMP1 model of chronic ileitis is unknown. Ten-week-old SAMP1 mice with AKR mice as control were treated with N6-(1-iminoethyl)-L-lysine dihydrochloride (L-NIL) to inhibit iNO production. Intracellular NO levels were found to be increased in villus cells from SAMP1 mice. Moreover, SGLT1 and Na+/K+-ATPase activities and BBM SGLT1 expression were significantly decreased. However, L-NIL treatment reduced the intracellular iNO production, and reversed both downregulated SGLT1 and Na+/K+-ATPase activities in SAMP1 mice. Inhibition of iNO by L-NIL treatment also significantly reversed the BBM SGLT1 protein expression in SAMP1 mice. L-NIL reversed the inflammation mediated downregulation of SGLT1 activity by restoring the BBM SGLT1 expression. Thus, regulation of SGLT1 in chronic ileitis is likely mediated by iNO.

Targeted removal of leukemia cells from the circulating system by whole-body magnetic hyperthermia in mice

Whole-body hyperthermia after intravenous injection of EpCAM antibody immobilized on magnetic nanoparticles (MNPs) decreased leukemia cells in AKR mice. Simulation analysis showed effective heat transfer from MNPs to leukemia cells.

P097 Depletion of delta-like ligand 4 (Dll4) contributes to the alteration of secretory progenitor differentiation in SAMP1/YitFc mice with Crohn’s disease-like ileitis

Background When the intestinal epithelium would be exposed to harmful substances, mucosal damage can be occurred and followed by epithelial restoration with regenerative epithelium derived from intestinal stem cells (ISCs). Crohn’s disease (CD) is characterised by chronic mucosal damage and ulceration predominantly involved in the ileum. Thus, we hypothesised that epithelial regeneration in CD might be impaired due to the dysfunction of ileal ISCs or its niche. Methods SAMP1/YitFcsI (SAMP1) mice are recombinant-inbred mice that spontaneously develop ileitis resembling human CD. Alterations in the crypt-villus structure, epithelial differentiation, organoid formation ability, and niche signalling pathways in the ileum of 10- to 14-week-old SAMP1 mice were analysed and compared with age-matched AKR mice. Results In the ileum of SAMP1 mice, the depth of the crypts increased and the surface area of the villi decreased. While the number of ISCs in the ileal crypts did not differ between SAMP1 and AKR mice. The number of Paneth cells decreased and the number of transient amplifying cells increased. The organoid formation ability of the ileal crypts of SAMP1 mice decreased significantly compared with that of AKR mice. RNA-seq revealed a differential gene expression pattern of ileal crypts of SAMP1 mice compared with those of AKR mice. Among the ISCs and niche signalling, the expression of delta-like ligand 4 (Dll4) was significantly decreased and the expression of genes associated with Notch signalling tended to decrease in the ileal crypts of SAMP1 mice. Conclusion The characteristic ileitis in SAMP1 mice may be caused by impaired Dll4-Notch signalling.

Xenotropic Mouse Gammaretroviruses Isolated from Pre-Leukemic Tissues Include a Recombinant

Naturally-occurring lymphomagenesis is induced by mouse leukemia viruses (MLVs) carried as endogenous retroviruses (ERVs). Replicating the ecotropic MLVs recombines with polytropic (P-ERVs) and xenotropic ERVs (X-ERVs) to generate pathogenic viruses with an altered host range. While most recovered nonecotropic recombinants have a polytropic host range, the X-MLVs are also present in the pre-leukemic tissues. We analyzed two such isolates from the AKR mice to identify their ERV progenitors and to look for evidence of recombination. AKR40 resembles the active X-ERV Bxv1, while AKR6 has a Bxv1-like backbone with substitutions that alter the long terminal repeat (LTR) enhancer and the envelope (env). AKR6 has a modified xenotropic host range, and its Env residue changes all lie outside of the domain that governs the receptor choice. The AKR6 segment spanning the two substitutions, but not the entire AKR6 env-LTR, exists as an ERV, termed Xmv67, in AKR, but not in the C57BL/6 mice. This suggests that AKR6 is the product of one, not two, recombination events. Xmv67 originated in the Asian mice. These data indicate that the recombinant X-MLVs that can be generated during lymphomagenesis, describe a novel X-ERV subtype found in the AKR genome, but not in the C57BL/6 reference genome, and identify residues in the envelope C-terminus that may influence the host range.

Double-hit mouse model of cigarette smoke priming for acute lung injury

Epidemiological studies indicate that cigarette smoking (CS) increases the risk and severity of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). The mechanism is not understood, at least in part because of lack of animal models that reproduce the key features of the CS priming process. In this study, using two strains of mice, we characterized a double-hit mouse model of ALI induced by CS priming of injury caused by lipopolysaccharide (LPS). C57BL/6 and AKR mice were preexposed to CS briefly (3 h) or subacutely (3 wk) before intratracheal instillation of LPS and ALI was assessed 18 h after LPS administration by measuring lung static compliance, lung edema, vascular permeability, inflammation, and alveolar apoptosis. We found that as little as 3 h of exposure to CS enhanced LPS-induced ALI in both strains of mice. Similar exacerbating effects were observed after 3 wk of preexposure to CS. However, there was a strain difference in susceptibility to CS priming for ALI, with a greater effect in AKR mice. The key features we observed suggest that 3 wk of CS preexposure of AKR mice is a reproducible, clinically relevant animal model that is useful for studying mechanisms and treatment of CS priming for a second-hit-induced ALI. Our data also support the concept that increased susceptibility to ALI/ARDS is an important adverse health consequence of CS exposure that needs to be taken into consideration when treating critically ill individuals.

Abstract 284: Cholesterol Handling in Bone Marrow--Derived Macrophages from Apoe-Deficient AKR and DBA/2 Mice

It has been demonstrated in humans that about 50% of the susceptibility to atherosclerosis is heritable. Therefore, it is not surprising that mice from distinct genetic backgrounds present different susceptibility to atherosclerosis. For example, apoE -/- DBA/2 mice have aortic root atherosclerosis lesion areas >10-fold larger than apoE -/- AKR mice. In order to better understand the mechanisms underlying this difference, we studied cholesterol metabolism in bone marrow-derived macrophages from these two strains. In the unloaded state, macrophages from both strains had equivalent amounts of total, esterified, and free cholesterol (TC, CE, and FC, respectively). Cells were loaded with acetylated low density lipoproteins (AcLDL) for 48h and DBA/2 macrophages had 36% higher TC (p<0.0001) vs. AKR macrophages, mainly due to 72% higher CE accumulation (p<0.0001). In contrast the loaded DBA/2 macrophages had 6% lower FC than the AKR macrophages (p<0.05). No difference was seen in AcLDL uptake or acetyl-coenzyme A acyltransferase (ACAT) activity between the two strains. When cells were loaded with AcLDL, the DBA/2 cells released cholesterol less efficiently than the AKR cells to apoAI (18% lower, p=0.01) or HDL (25% lower, p=0.001). However when the loading was performed in presence of ACAT inhibitor, blocking the formation of CE, the efflux from the two strains was equivalent suggesting a blockage of CE hydrolysis in DBA/2 cells. In loaded cells, when a 24h chase period was done in presence of ACAT inhibitor to prevent cholesterol re-esterification, the CE was reduce by 69% in AKR cells (t 1/2 = 47h) compared to chase without inhibitor while it was only reduced by 42% in DBA/2 cells (t 1/2 = 101h). Furthermore, when lalistat, an inhibitor of lysosomal acid lipase (LAL), and ACAT inhibitor were added during the chase, CE levels were equivalent to the one observed in chase without any inhibitors, suggesting that CE hydrolysis occurs primarily in the lysosome. In conclusion, our data support the hypothesis that DBA/2 cells accumulate more CE than AKR cells due to a defect in CE hydrolysis by LAL. Autophagy has been recently described as the pathway in macrophages by which lipid droplets can be delivered to the lysosome, and we are investigating the role of this pathway in our cells.

Spontaneous autoimmune gastritis and hypochlorhydria are manifest in the ileitis-prone SAMP1/YitFcs mice

SAMP1/YitFcs mice serve as a model of Crohn's disease, and we have used them to assess gastritis. Gastritis was compared in SAMP1/YitFcs, AKR, and C57BL/6 mice by histology, immunohistochemistry, and flow cytometry. Gastric acid secretion was measured in ligated stomachs, while anti-parietal cell antibodies were assayed by immunofluorescence and enzyme-linked immunosorbent spot assay. SAMP1/YitFcs mice display a corpus-dominant, chronic gastritis with multifocal aggregates of mononuclear cells consisting of T and B lymphocytes. Relatively few aggregates were observed elsewhere in the stomach. The infiltrates in the oxyntic mucosa were associated with the loss of parietal cell mass. AKR mice, the founder strain of the SAMP1/YitFcs, also have gastritis, although they do not develop ileitis. Genetic studies using SAMP1/YitFcs-C57BL/6 congenic mice showed that the genetic regions regulating ileitis had comparable effects on gastritis. The majority of the cells in the aggregates expressed the T cell marker CD3 or the B cell marker B220. Adoptive transfer of SAMP1/YitFcs CD4+ T helper cells, with or without B cells, into immunodeficient recipients induced a pangastritis and duodenitis. SAMP1/YitFcs and AKR mice manifest hypochlorhydria and anti-parietal cell antibodies. These data suggest that common genetic factors controlling gastroenteric disease in SAMP1/YitFcs mice regulate distinct pathogenic mechanisms causing inflammation in separate sites within the digestive tract.

Mechanisms underlying gut dysfunction in a murine model of chronic parasitic infection

Irritable bowel syndrome (IBS) is common in countries where chronic parasitic infestations are endemic. However, the relationship between parasitic infection and IBS is not clear. The aim of this study was to examine whether chronic parasitic infection is accompanied by gut dysfunction and whether the continued presence of the parasite is required for the maintenance of the dysfunction. We used chronic Trichuris muris infection in Th1-biased susceptible AKR mice to evaluate this relationship. AKR mice were infected with T. muris and were euthanized on various days postinfection (pi) to examine worm burden, muscle function, and immune and inflammatory responses. Mice were treated with the anthelmintic oxantel pamoate to assess the effect of eradication of infection on muscle function. Infection resulted in persistence of the parasite, elevated IFN-γ, and increased MPO activity evident at 45 days pi. This was accompanied by a reduction in muscle contractility and excitatory innervation. Whereas parasite eradication at 7 days pi normalized IFN-γ and muscle contractility, eradication at 28 days pi failed to normalize muscle contractility. Administration of dexamethasone after parasite eradication normalized all parameters. Anthelmintic treatment improved histology except for eosinophils, which were normalized by subsequent dexamethasone therapy. Persistent gut dysfunction is independent of the continued presence of the parasite and is maintained by inflammatory process that includes eosinophils. Thus data in this preclinical model suggest that parasitic infection could be a cause of IBS, and the lack of symptomatic improvement following eradication is insufficient evidence to refute a causal relationship between the infection and IBS.