DSS-treated Wild-type Research Articles

PTK2B regulates immune responses of neutrophils and protects mucosal inflammation in ulcerative colitis.

Neutrophils participate in the pathogenesis of ulcerative colitis (UC) through regulating the intestinal homeostasis. Several inflammatory diseases are reported to be regulated by proline-rich tyrosine kinase 2B (PTK2B). However, the role of PTK2B in regulating the function of neutrophils and the pathogenesis of UC remains unknown. In this study, the mRNA and protein levels of PTK2B in the colonic tissues from UC patients were measured by using quantitative real-time polymerase chain reaction (qRT-PCR), western blotting, and immunohistochemistry. TAE226, a PTK2B inhibitor, was used to inhibit the activity of PTK2B in neutrophils, and then, the pro-inflammatory factors were analyzed by using qRT-PCR and ELISA. To determine the role of PTK2B in intestinal inflammation, a dextran sulfate sodium (DSS)-induced colitis model was established in PTK2B gene knockout (PTK2B KO) and wild-type (WT) mice. We found that compared with healthy donor controls, the expression level of PTK2B was significantly elevated in inflamed mucosa from UC patients. In addition, expression of PTK2B was positively correlated with the severity of disease. Pharmacological inhibition of PTK2B could markedly reduce the generation of reactive oxygen species (ROS), myeloperoxidase (MPO), and antimicrobial peptides (S100a8 and S100a9) in neutrophils. The vitro study showed that tumor necrosis factor (TNF)-α is involved in promoting the expression of PTK2B in neutrophils. As expected, UC patients treated with infliximab, an anti-TNF-α agent, showed significantly reduced level of PTK2B in neutrophils, as well as in the intestinal mucosa. Of note, compared with DSS-treated WT mice, DSS-treated PTK2B KO mice showed more severe colitis symptoms. Mechanistically, PTK2B could enhance neutrophil migration by regulating CXCR2 and GRK2 expression via the p38 MAPK pathway. Additionally, mice treated with TAE226 exhibited the same effects. In conclusion, PTK2B is involved in the pathogenesis of UC by promoting the migration of neutrophils and inhibiting mucosal inflammation, highlighting PTK2B as a new potential therapeutic target to treat UC.

A Circulating Microbial Metabolite Drives the Clonal Expansion of Pre-Leukemic Cells

Inflammatory mediators, including cytokines and microbial ligands, are implicated in the development of myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). A consequence of aging and various autoimmune and inflammatory conditions is loss of intestinal barrier integrity and translocation of microbial species and metabolites into circulation. More recent findings revealed that patients with inflammatory bowel disease (IBD) harbor mutant hematopoietic clones and often present with clinical manifestations of MDS/AML. Moreover, intestinal barrier dysfunction is commonly reported at diagnosis in patients with MDS and AML. These observations led us to speculate that microbial-derived signals may contribute to the expansion of pre-leukemic hematopoietic stem and progenitor cells (HSPCs) and development of myeloid malignancies. To determine if loss of intestinal barrier integrity contributes to the expansion of pre-leukemic DNMT3a-mutant HSPCs, we generated a model of IBD by inducing gut epithelial injury using dextran sulfate sodium (DSS). Upon gut epithelial injury, we observed a significant expansion of DNMT3a-mutant HSPCs, but not of wild-type (WT) HSPCs, in vivo. Unexpectedly, DNMT3a-mutant HSPCs do not exhibit a competitive advantage over WT cells in the absence of intestinal barrier dysfunction, such as following DSS or high-dose radiation. Fecal material from DSS-treated WT mice was sufficient to induce expansion of DNMT3a-mutant HSPCs and this expansion was prevented with broad-spectrum antibiotics. These findings indicate that the competitive advantage of DNMT3a-mutant HSPCs following gut epithelial injury was due to alterations in the microbiome. In parallel sequencing studies, we identified a significant enrichment of gram-negative bacterial phyla in stool samples from the IBD mouse model, IBD patients, and treatment-naive AML patients. All gram-negative bacteria uniquely generate a microbial intermediate, ADP-heptose, as part of the lipopolysaccharide biosynthesis pathway. ADP-heptose can enter the blood circulation and freely translocate into cells and activate the ALPK1-TIFA complex ("TIFAsome"). Alpha-protein kinase 1 (ALPK1) and tumor necrosis factor receptor-associated factor (TRAF)-interacting protein with an FHA domain (TIFA) are overexpressed in primary human MDS and AML HSPCs as compared to healthy donors and their expression is correlated with poor prognosis. Gram negative bacteria and corresponding ADP-heptose levels were also significantly increased in the plasma of individuals with intestinal barrier injury, such with IBD, MDS, or upon aging, as compared to young healthy individuals. The circulating levels of ADP-heptose from aged individuals and IBD or MDS patients were sufficient to induce the TIFAsome in leukemic cells. Moreover, administration of disease relevant levels of ADP-heptose resulted in expansion and enhanced function of mouse DNMT3a-mutant HSPCs and of human AML cell lines and primary MDS cells in vivo. Expansion of DNMT3a-mutant HSPCs and human MDS/AML cells was directly related to overexpression of ALPK1, as WT HSPCs or ALPK1-deficient AML cells did not respond to ADP-heptose. To gain deeper mechanistic insight, we examined global transcriptional and epigenetic changes in purified WT and DNTM3a-mutant HSCs treated with ADP-heptose. DNMT3a-mutant HSCs treated with ADP-heptose resulted in epigenetic changes and increased expression of gene programs related to non-canonical NF-kB, ERK/MAPK, cell cycle activation, cell survival, and stem cell function. Indeed, ADP-heptose caused increased cycling and self-renewal of DNMT3a-mutant HSPCs, but not WT HSPCs, via the TIFAsome in vitro and in vivo. Based on the enriched pathways in DNMT3a-mutant HSCs treated with ADP-heptose, we screened small molecule inhibitors of TIFAsome activation. We found that targeting UBE2N abrogated ADP-heptose-mediated signaling via the TIFAsome and expansion of patient-derived AML xenografts. In summary, we report that conditions leading to intestinal barrier dysfunction and circulating ADP-heptose from gram-negative bacteria induce expansion of pre-leukemic and MDS/AML cells via ALPK1-TIFA. Lastly, we provide a potential therapeutic strategy to prevent the competitive advantage of pre-leukemic cells prior to overt malignant transformation.

Blocking DCIR mitigates colitis and prevents colorectal tumors by enhancing the GM-CSF-STAT5 pathway.

Dendritic cell immunoreceptor (DCIR; Clec4a2), a member of the C-type lectin receptor family, plays important roles in homeostasis of the immune and bone systems. However, the intestinal role of this molecule is unclear. Here, we show that dextran sodium sulfate (DSS)-induced colitis and azoxymethane-DSS-induced intestinal tumors are reduced in Clec4a2-/- mice independently of intestinal microbiota. STAT5 phosphorylation and expression of Csf2 and tight junction genes are enhanced, while Il17a and Cxcl2 are suppressed in the Clec4a2-/- mouse colon, which exhibits reduced infiltration of neutrophils and myeloid-derived suppressor cells. Granulocyte-macrophage colony-stimulating factor (GM-CSF) administration ameliorates DSS colitis associated with reduced Il17a and enhanced tight junction gene expression, whereas anti-GM-CSF exacerbates symptoms. Furthermore, anti-NA2, a ligand for DCIR, ameliorates colitis and prevents colorectal tumors. These observations indicate that blocking DCIR signaling ameliorates colitis and suppresses colonic tumors, suggesting DCIR as a possible target for the treatment of these diseases.

Study on the additive protective effect of PGLYRP3 and Bifidobacterium adolescentis Reuter 1963 on severity of DSS-induced colitis in Pglyrp3 knockout (Pglyrp3 −/−) and wild-type (WT) mice

Background and AimsPglyrp3 is a bactericidal innate immunity protein known to sustain the habitual gut microbiome and protect against experimental colitis. Intestinal inflammation and metaflammation are commonly associated with a marked reduction of commensal bifidobacteria. Whether Pglyrp3 and bifidobacteria interact synergistically or additively to alleviate metaflammation is unknown. We investigated the extent to which Pglyrp3 and bifidobacteria regulate metaflammation and gut bacterial dysbiosis in DSS-induced mouse models of intestinal inflammation. Material & Methods8–10 weeks old male mice were used. In both WT and Pglyrp3 −/− experiments, the mice were randomly divided into three groups of 16 mice per group: (1) a control group receiving sterile tap water, (2) an experimental group receiving sterile tap water supplemented with only 5% DSS, and (3) an experimental group receiving sterile tap water supplemented with 5% DSS and 1 × 109 CFU/ml of Bifidobacterium adolescentis (B.a.) for 7 days. Wild-type (WT) littermates of the respective gene (i.e. Pglyrp3) were used as controls throughout the study. Clinical signs of general health and inflammation were monitored daily. Faecal pellet samples were analysed by qRT-PCR for microbial composition. Histology of relevant organs was carried out on day 8. Metabolic parameters and liver inflammation were determined in serum samples. ResultsIntestinal inflammation in mice of group 2 were significantly increased compared to those of control group 1. There was a significant difference in mean scores for inflammation severity between DSS-treated WT and DSS-treated Pglyrp3 −/− mice. Buildup of key serum metabolic markers (cholesterol, triglyceride and glucose) was set off by colonic inflammation. qRT-PCR quantification showed that DSS significantly decreased the Clostridium coccoides and Bifidobacterium cell counts while increasing those of Bacteroides group in both WT and Pglyrp3 −/− mice. These manifestations of DSS-induced dysbiosis were significantly attenuated by feeding B.a. Both the local and systemic ill-being of the mice alleviated when they received B.a. DiscussionThis study shows that Pglyrp3 facilitates recognition of bifidobacterial cell wall-derived peptidoglycan, thus leading additively to a reduction of metaflammation through an increase in the number of bifidobacteria, which were able to mitigate intestinal immunopathology in the context of Pglyrp3 blockade.

Mice Deficient in Cyp4a14 Have An Increased Number of Goblet Cells and Attenuated Dextran Sulfate Sodium-Induced Colitis

Background/Aims: Cyp4a14 is a member of cytochrome P450 (Cyp450) enzyme superfamily that possesses NADPH monooxygenase activity, which catalyzes omega-hydroxylation of medium-chain fatty acids and arachidonic acid. Study suggests that down-regulation of Cyp4a14 has an anti-inflammatory response in intestine. The present study was to test the function of Cyp4a14 in dextran sulfate sodium (DSS)-induced colitis. Methods: Female Cyp4a14-knockout (KO) and wild-type (WT) mice were treated with DSS for 6 days to induce colitis. The colon of mice was histologically observed by hematoxylin and eosin (H&E) and periodic acid Schiff (PAS) staining. The serum malondialdehyde (MDA), an endogenous indicator of oxidative stress, was chemically measured. Proinflammatory and NADPH oxidase genes were examined by quantitative polymerase chain reaction (qPCR). Results: Cyp4a14-KO mice had a significantly higher number of goblet cells in the colon and were more resistant to DSS-induced colitis compared with the WT mice. The DSS-treated KO mice had lower levels of MDA. Consistent with the milder inflammatory pathological changes, DSS-treated KO mice had lower levels of IL-1β, IL-6 and TNF-α mRNA in the liver and the colon. Moreover, the colon of DSS-treated Cyp4a14-KO and WT mice had higher mRNA levels of two members of NADPH oxidases, Nox2 and Nox4, suggesting that both Nox2 and Nox4 are inflammatory markers. By contrast, DSS-treated WT and KO mice had drastically decreased epithelium-localized Nox1 and dual oxidase (Duox) 2 mRNA levels, coinciding with the erosion of the mucosa induced by DSS. Conclusion: These results suggests a hypothesis that the increased goblet cell in the colon of Cyp4a14-KO mice provides protection from mucosal injury and Cyp4a14-increased oxidative stress exacerbates DSS-induced colitis. Therefore, Cyp4a14 may represent a potential target for treating colitis.

A key requirement for CD300f in innate immune responses of eosinophils in colitis.

Eosinophils are traditionally studied in the context of type 2 immune responses. However, recent studies highlight key innate immune functions for eosinophils especially in colonic inflammation. Surprisingly, molecular pathways regulating innate immune activities of eosinophil are largely unknown. We have recently shown that the CD300f is highly expressed by colonic eosinophils. Nonetheless, the role of CD300f in governing innate immune eosinophil activities is ill-defined. RNA sequencing of 162 pediatric Crohn's disease patients revealed upregulation of multiple Cd300 family members, which correlated with the presence of severe ulcerations and inflammation. Increased expression of CD300 family receptors was also observed in active ulcerative colitis (UC) and in mice following induction of experimental colitis. Specifically, the expression of CD300f was dynamically regulated in monocytes and eosinophils. Dextran sodium sulfate (DSS)-treated Cd300f-/- mice exhibit attenuated disease activity and histopathology in comparison with DSS-treated wild type (WT). Decreased disease activity in Cd300f-/- mice was accompanied with reduced inflammatory cell infiltration and nearly abolished production of pro-inflammatory cytokines. Monocyte depletion and chimeric bone marrow transfer experiments revealed a cell-specific requirement for CD300f in innate immune activation of eosinophils. Collectively, we uncover a new pathway regulating innate immune activities of eosinophils, a finding with significant implications in eosinophil-associated gastrointestinal diseases.

Mast Cells Infiltrating Inflamed or Transformed Gut Alternatively Sustain Mucosal Healing or Tumor Growth.

Mast cells (MC) are immune cells located next to the intestinal epithelium with regulatory function in maintaining the homeostasis of the mucosal barrier. We have investigated MC activities in colon inflammation and cancer in mice either wild-type (WT) or MC-deficient (Kit(W-sh)) reconstituted or not with bone marrow-derived MCs. Colitis was chemically induced with dextran sodium sulfate (DSS). Tumors were induced by administering azoxymethane (AOM) intraperitoneally before DSS. Following DSS withdrawal, Kit(W-sh) mice showed reduced weight gain and impaired tissue repair compared with their WT littermates or Kit(W-sh) mice reconstituted with bone marrow-derived MCs. MCs were localized in areas of mucosal healing rather than damaged areas where they degraded IL33, an alarmin released by epithelial cells during tissue damage. Kit(W-sh) mice reconstituted with MC deficient for mouse mast cell protease 4 did not restore normal mucosal healing or reduce efficiently inflammation after DSS withdrawal. In contrast with MCs recruited during inflammation-associated wound healing, MCs adjacent to transformed epithelial cells acquired a protumorigenic profile. In AOM- and DSS-treated WT mice, high MC density correlated with high-grade carcinomas. In similarly treated Kit(W-sh) mice, tumors were less extended and displayed lower histologic grade. Our results indicate that the interaction of MCs with epithelial cells is dependent on the inflammatory stage, and on the activation of the tissue repair program. Selective targeting of MCs for prevention or treatment of inflammation-associated colon cancer should be timely pondered to allow tissue repair at premalignant stages or to reduce aggressiveness at the tumor stage.

Intestinal epithelium-specific knockout of the cytochrome P450 reductase gene exacerbates dextran sulfate sodium-induced colitis.

The potential involvement of intestinal microsomal cytochrome P450 (P450) enzymes in defending against colon inflammation and injury was studied in mice treated with dextran sulfate sodium (DSS) to induce colitis. Wild-type (WT) mice and mice with intestinal epithelium (IE)-specific deletion of the P450 reductase gene (IE-Cpr-null) were compared. IE-Cpr-null mice have little microsomal P450 activity in IE cells. DSS treatment (2.5% in drinking water for 6 days) caused more severe colon inflammation, as evidenced by the presence of higher levels of myeloperoxidase and proinflammatory cytokines [tumor necrosis factor-α, interleukin (IL)-6, and IL-1β], and greater weight loss, colonic tissue damage, and colon shortening, in IE-Cpr-null mice than in WT mice. The IE-Cpr-null mice were deficient in colonic corticosterone (CC) synthesis, as indicated by the inability of ex vivo cultured colonic tissues from DSS-treated IE-Cpr-null mice (in contrast to DSS-treated WT mice) to show increased CC production, compared with vehicle-treated mice, and by the ability of added deoxycorticosterone (DOC), a precursor of CC biosynthesis via mitochondrial CYP11B1, to restore ex vivo CC production by colonic tissues from DSS-treated null mice. Intriguingly, null (but not WT) mice failed to show increased serum CC levels following DSS treatment. Nevertheless, cotreatment of DSS-exposed mice with DOC, which did not restore DSS-induced increase in serum CC, abolished the hypersensitivity of IE-Cpr-null mice to DSS-induced colon injury. Taken together, our results strongly support the notion that microsomal P450 enzymes in the intestine play an important role in protecting colon epithelium from DSS-induced inflammation and injury, possibly through increased local CC synthesis in response to DSS challenge.

PepT1 expressed in immune cells has an important role in promoting the immune response during experimentally induced colitis

We and others have shown that the dipeptide cotransporter PepT1 is expressed in immune cells, including macrophages that are in close contact with the lamina propria of the small and large intestines. In the present study, we used PepT1-knockout (KO) mice to explore the role played by PepT1 in immune cells during dextran sodium sulfate (DSS)-induced colitis. DSS treatment caused less severe body weight loss, diminished rectal bleeding, and less diarrhea in PepT1-KO mice than in wild-type (WT) animals. A histological examination of colonic sections revealed that the colonic architecture was less disrupted and the extent of immune cell infiltration into the mucosa and submucosa following DSS treatment was reduced in PepT1-KO mice compared with WT animals. Consistent with these results, the DSS-induced colitis increase in colonic myeloperoxidase activity was significantly less in PepT1-KO mice than in WT littermates. The colonic levels of mRNAs encoding the inflammatory cytokines CXCL1, interleukin (IL)-6, monocyte chemotactic protein-1, IL-12, and interferon-γ were significantly lower in DSS-treated PepT1-KO mice than in DSS-treated WT animals. Colonic immune cells from WT had significantly higher level of proinflammatory cytokines then PepT1 KO. In addition, we observed that knocking down the PepT1 expression decreases chemotaxis of immune cells recruited during intestinal inflammation. Antibiotic treatment before DSS-induced colitis eliminated the differential expression of inflammatory cytokines between WT and PepT1-KO mice. In conclusion, PepT1 in immune cells regulates the secretion of proinflammatory cytokines triggered by bacteria and/or bacterial products, and thus has an important role in the induction of colitis. PepT1 may transport small bacterial products, such as muramyl dipeptide and the tripeptide L-Ala-gamma-D-Glu-meso-DAP, into macrophages. These materials may be sensed by members of the nucleotide-binding site–leucine-rich repeat family of intracellular receptors, ultimately resulting in altered homeostasis of the intestinal microbiota.

Exacerbation of DSS‐induced colitis in mice lacking kinin B1 receptors through compensatory up‐regulation of kinin B2 receptors: the role of tight junctions and intestinal homeostasis

Kinins are pro-inflammatory peptides that are released during tissue injury, including that caused by inflammatory bowel disease. Herein, we assessed the role and underlying mechanisms through which the absence of kinin B(1) receptors exacerbates the development of dextran sulfate sodium (DSS)-induced colitis in mice. B(1) and B(2) receptor antagonists and B(1) receptor knockout mice (B1(-/-) ) were used to assess the involvement of B(1) and B(2) receptor signalling in a DSS-colitis. B(1) receptor, B(2) receptor, occludin and claudin-4 expression, cytokine levels and cell permeability were evaluated in colon from wild-type (WT) and B1(-/-) mice. DSS-induced colitis was significantly exacerbated in B1(-/-) compared with WT mice. IL-1β, IFN-γ, keratinocyte-derived chemokine and macrophage inflammatory protein-2 were markedly increased in the colon from DSS-treated B1(-/-) compared with DSS-treated WT mice. Treatment of WT mice with a selective B(1) receptor antagonist, DALBK or SSR240612, had no effect on DSS-induced colitis. Of note, B(2) receptor mRNA expression was significantly up-regulated in colonic tissue from the B1(-/-) mice after DSS administration. Moreover, treatment with a selective B(2) receptor antagonist prevented the exacerbation of colitis in B1(-/-) mice following DSS administration. The water- or DSS-treated B1(-/-) mice showed a decrease in occludin gene expression, which was partially prevented by the B(2) receptor antagonist. A loss of B(1) receptors markedly exacerbates the severity of DSS-induced colitis in mice. The increased susceptibility of B1(-/-) may be associated with compensatory overexpression of B(2) receptors, which, in turn, modulates tight junction expression.

Abstract 4279: Inflammation increases colonic tumor multiplicity and stage in Pirc rats: A priming effect

Abstract Colon cancer is the second most common cancer in men and women in the United States. The average lifetime risk for developing colon cancer is about 7%. This lifetime risk is increased several fold in individuals with an inflammatory bowel disease, and increases further with greater extent, duration and severity of inflammation. The role of inflammation in many cancers is becoming clearer, but the mechanism(s) by which inflammation may initiate or support cancer growth remains to be elucidated. Our laboratory has developed a rodent model of colon cancer, the Polyposis in rat colon (Pirc) rat, which closely mimics the predisposition for colonic neoplasia observed in humans. Colonic inflammation in the Pirc rat induced by dextran sodium sulfate (DSS) increases the number of colon tumors up to ten-fold, and include invasive adenocarcinomas as early as five months of age. To ascertain molecular differences between colonic tumors and normal colonic epithelium in DSS-treated Pirc rats, and colonic tumors and normal colonic epithelium in untreated Pirc rats, tissues were harvested at 97 days of age, before any detectable difference in tumor invasion. Agilent Whole Rat Genome expression microarrays were run on several biological replicates of each sample type. As expected, many genes (4630) were differentially expressed between normal epithelium and colonic tumors (>2-fold change, 0.05 FDR). Surprisingly, no significant difference in gene expression was observed between tumors from untreated and DSS-treated Pirc rats. However, over 950 genes showed differential expression in normal epithelium between untreated and DSS-treated Pirc rats. Further, many of the genes in apparently normal epithelial tissue from DSS-treated Pirc rats showed an expression profile intermediate between normal untreated Pirc epithelium and tumors. Several of these candidates have been confirmed by qRT-PCR. DSS-treated animals eventually develop a large number of tumors throughout the colon. This expression profile could indicate that this apparently normal tissue contains nascent tumors. Alternatively, this could represent changes in the normal tissue surrounding the tumor that supports the growth and invasion of colonic neoplasms. This distinction requires further investigation using other biological endpoints. Wild type rats treated with DSS develop a “cobblestone” pattern in the colon, but rarely form even a single tumor. Microarray profiling of normal epithelium from untreated and DSS-treated wild type rats may reveal whether the signatures we have seen in the apparently normal colonic epithelium from DSS-treated Pirc rats is simply an undetected tumor. Using endoscopy we can longitudinally obtain biopsy samples to see whether these expression profiles change over time from normal epithelium to an invasive cancer. Comparing our data to that in humans with ulcerative colitis will help focus future exploration of candidate genes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4279. doi:1538-7445.AM2012-4279

Characterization of the Cysteinyl Leukotriene 2 Receptor in Novel Expression Sites of the Gastrointestinal Tract

Cysteinyl leukotrienes (cysLTs: LTC₄, LTD₄, and LTE₄) are pro-inflammatory lipid molecules synthesized from arachidonic acid. They exert their actions on at least two cysLT receptors (CysLT₁R and CysLT₂R). Endothelial expression and activation of these receptors is linked to vasoactive responses and to the promotion of vascular permeability. Here we track the expression pattern of CysLT₂R in a loss-of-function murine model (CysLT₂R-LacZ) to neurons of the myenteric and submucosal plexus in the small intestine, colonic myenteric plexus, dorsal root ganglia, and nodose ganglion. Cysteinyl leukotriene (LTC₄/D₄) stimulation of colonic submucosal venules elicited a greater permeability response in wild-type mice. In a dextran sulfate sodium-induced colon inflammation model, the disease activity index and colonic edema (measured by wet:dry weights and submucosal thickness) were significantly reduced in knockout (KO) mice compared to controls. Tumor necrosis factor-α levels in colon tissue were significantly lower in KO mice; however, myeloperoxidase activity was similar in both the KO and wild-type groups. Finally, patch-clamp recordings of basal neuronal activity of colonic-projecting nociceptive neurons from dorsal root ganglia (T9-13) revealed significantly higher excitability in KO neurons compared to wild type. These results suggest that a lack of neuronal expression of CysLT₂R in the murine colonic myenteric plexus attenuates colitis disease progression via a reduction in inflammation-associated tissue edema and increases neuronal sensitivity to nociceptive stimuli.

Knock-Out of β-Glucosidase 2 Has No Influence on Dextran Sulfate Sodium-Induced Colitis

Background/Aims: The non-lysosomal glucosylceramidase, β-glucosidase (Gba2), hydrolyzes glucosylceramide to glucose and ceramide (Cer). Cer is a potent second-messenger lipid that plays an important role in signaling cascades involved in apoptosis. The aim of this study was to investigate whether Gba2 knock-out (Gba2^–/–) affects the extent of dextran sulfate sodium (DSS)-induced colitis in mice. Methods: Acute colitis was induced in wild-type (WT) and Gba2^–/– mice by administration of 2% DSS in drinking water. After 7 days, mice underwent colonoscopy and were sacrificed. Results: Both DSS-treated WT (n = 10) and Gba2^–/– (n = 12) mice showed elevated histological and endoscopic scores compared to respective H₂O controls (n = 9 each). However, no significant differences between the DSS groups were detected. Flow cytometric analysis of propidium iodide staining, cleavage of caspases-3 and -8, indicative for apoptosis, as well as Cer levels were not altered in DSS-treated WT or Gba2^–/– mice. Gba2^–/– resulted in slightly decreased expression of glucocerebrosidase (Gba1) as well as in upregulation of proteins being involved in cellular regeneration, such as STAT3 (signal transducer and activator of transcription), JNK and iNOS, upon DSS treatment. Conclusion: We demonstrate that Gba2^–/– does not affect the extent of DSS-induced inflammation in mice, however, it might be involved in tissue regeneration in response to toxic agents.

Intestinal alkaline phosphatase has beneficial effects in mouse models of chronic colitis

The brush border enzyme intestinal alkaline phosphatase (IAP) functions as a gut mucosal defense factor and is protective against dextran sulfate sodium (DSS)-induced acute injury in rats. The present study evaluated the potential therapeutic role for orally administered calf IAP (cIAP) in two independent mouse models of chronic colitis: 1) DSS-induced chronic colitis, and 2) chronic spontaneous colitis in Wiskott-Aldrich Syndrome protein (WASP)-deficient (knockout) mice that is accelerated by irradiation. The wildtype (WT) and IAP knockout (IAP-KO) mice received four cycles of 2% DSS ad libitum for 7 days. Each cycle was followed by a 7-day DSS-free interval during which mice received either cIAP or vehicle in the drinking water. The WASP-KO mice received either vehicle or cIAP for 6 weeks beginning on the day of irradiation. Microscopic colitis scores of DSS-treated IAP-KO mice were higher than DSS-treated WT mice (52±3.8 versus 28.8±6.6, respectively, P<0.0001). cIAP treatment attenuated the disease in both groups (KO=30.7±6.01, WT=18.7±5.0, P<0.05). In irradiated WASP-KO mice cIAP also attenuated colitis compared to control groups (3.3±0.52 versus 6.2±0.34, respectively, P<0.001). Tissue myeloperoxidase activity and proinflammatory cytokines were significantly decreased by cIAP treatment. Endogenous IAP appears to play a role in protecting the host against chronic colitis. Orally administered cIAP exerts a protective effect in two independent mouse models of chronic colitis and may represent a novel therapy for human IBD.

Mast cell-restricted Tryptases are essential in a Mouse Model of Inflammatory Bowel Disease

Patients with inflammatory bowel disease (IBD) often display increased numbers of colonic hTryptase-β1+ mast cells (MCs), but the biologic significance of this observation has not been elucidated. Mouse mast cell protease-6 (mMCP-6) is the ortholog of hTryptase-β1, and we previously showed that this tetramer- forming tryptase has beneficial roles in bacterial and helminth infections but adverse roles in arthritis. Due to the latter findings, we hypothesized that mMCP-6 might have comparable pro-inflammatory and/or connective tissue remodeling roles in experimental colitis. mMCP-6-null C57BL/6 (B6) mice were evaluated in the dextran sodium sulfate (DSS) experimental colitis model. These and age-matched wild-type (WT) B6 mice were given 1.5-2.0% DSS in their drinking water for 7 days, and the extent of weight loss was assessed, as well as the individual colon histopathology scores. RNA was isolated, and the levels of the transcripts that correspond to all known mouse proteins in the diseased colons were accessed by microarray analysis. Confirmatory real-time qPCR, ELISA, and/or immunohistochemistry analyses were carried out on a number of the induced transcripts that encode those cytokines, chemokines, and matrix metalloproteinases (MMPs) previously implicated in IBD. DSS-treated WT mice lost 7.01% ± 1.02 (n = 27) of their original weight, whereas similarly treated mMCP-6-null mice did not lose weight (0.21%±1.02, n= 26, p<0.0001). Using a 20-point histopathology scoring system, WT mice scored a more severe 12.33 ± 0.75 relative to similarly-treated mMCP-6-null mice which scored 5.00 ± 0.99 (p<0.0001). GeneChip analysis and real-time qPCR analysis of extracted RNA from distal colon segments revealed that numerous pro-inflammatory cytokines [e.g. IL-1β, IL-6, and IL-13), chemokines that preferentially attract neutrophils (e.g., Cxcl1, Cxcl2, and Cxcl3), and MMPs that participate in the remodeling of extracellular matrices (e.g., MMP3, MMP9, and MMP13) were all markedly increased in the colons of DSS-treated WT mice relative to untreated WT mice and DSS-treated mMCP-6-null mice. ELISA and/or immunohistochemistry analyses revealed changes in the corresponding protein levels of many of these differentially expressed transcripts. mMCP-6 is a critical mediator in the DSS model of murine experimental colitis, and this tryptase acts upstream of many of those factors implicated in IBD. The accumulated data raise the possibility that mast cell hTryptase-β1 has prominent adverse roles in patients with IBD.

Osteopontin attenuation of dextran sulfate sodium-induced colitis in mice

Osteopontin (OPN) is a matricellular cytokine present in most tissues and body fluids; it is known to modulate immune responses. In previous studies using the dextran sulfate sodium (DSS) acute colitis model, we found exacerbated tissue destruction and reduced repair in OPN-null (−/−) mice compared with wild-type (WT) controls. As OPN is normally present in milk, we hypothesized that administration of OPN may protect the intestines from the adverse effects of experimental colitis. A volume of 20 or 2 μg/ml bovine milk OPN, dissolved in drinking water, was given to mice 24 h before, and during administration of DSS. Clinical parameters of colitis and neutrophil functions were analyzed as previously reported. Orally administered OPN was absorbed and detected in the colon mucosa by immunohistochemistry. The 20 μg/ml OPN- and DSS-treated WT mice showed 37% less weight loss and reduced colon shortening and spleen enlargements than control mice (P<0.05). OPN administration also reduced the disease activity index, improved red blood cell counts, and reduced gut neutrophil activity compared with the DSS-treated WT mice that were not administered OPN (P<0.05). Immunohistochemical detection of F4/80-labelled cells (macrophages) was also less frequent. The level of transforming growth factor β1 (TGF-β1) was increased and the levels of pro-inflammatory mediators decreased in colon tissue samples of OPN-treated mice analyzed by ELISA. The reversal of experimental colitis parameters by exogenous OPN was not as robust in the OPN−/− mice. Administration of prokaryotic-expressed recombinant OPN and bovine serum albumin were ineffective. This study shows that administration of a physiological concentration of milk OPN in drinking water ameliorates the destructive host response in DSS-induced acute colitis.

Gene expression patterns in experimental colitis in IL-10-deficient mice

While others have described gene expression patterns in humans with inflammatory bowel diseases and animals with chemically induced colitis, a genome-wide comparison of gene expression in genetically susceptible animals that develop spontaneous colitis has not been reported. We used microarray technology to compare gene expression profiles in cecal specimens from specific pathogen-free IL10-deficient (IL10-/-) mice with colitis and normal wildtype (WT) mice. RNA isolated from ceca of IL10-/- and WT mice was subjected to microarray analysis. The results were confirmed by real-time polymerase chain reaction (PCR) and immunofluorescence microscopy of selected molecules. Expression of the selected genes in dextran sodium sulfate (DSS)-treated mice with colitis and epithelial cell lines activated with pathophysiologic stimuli was measured by real-time PCR. Histological inflammation of the colon and IL-12/23p40 secretion from intestinal explants were greater in IL10-/- and DSS-treated mice versus WT and untreated mice. Microarray analysis demonstrated >10-fold induction of the following molecules in the ceca of IL10-/- mice: mitochondrial ribosomal protein-L33, aquaporin-4, indoleamine-pyrrole-2,3-dioxygenase, and MHC class II with 63, 25, 20, and 12-fold increases, respectively. Cytochrome-P450, pancreatic lipase-related protein-2, and transthyretin were downregulated in IL10-/- mice. MHC II was increased throughout the colon, and aquaporin-4 was increased in the basolateral aspect of cecal epithelial cells. MHC II mRNA was increased in epithelial cells treated with IFN-gamma, but not TNF or Toll-like receptor ligands. Although most upregulated genes in experimental colitis are immune-related, aquaporin-4 and mitochondrial ribosomal protein-L33, which have not been previously associated with inflammation, were most highly upregulated.

Annexin A1 Regulates Intestinal Mucosal Injury, Inflammation, and Repair

During mucosal inflammation, a complex array of proinflammatory and protective mechanisms regulates inflammation and severity of injury. Secretion of anti-inflammatory mediators is a mechanism that is critical in controlling inflammatory responses and promoting epithelial restitution and barrier recovery. AnxA1 is a potent anti-inflammatory protein that has been implicated to play a critical immune regulatory role in models of inflammation. Although AnxA1 has been shown to be secreted in intestinal mucosal tissues during inflammation, its potential role in modulating the injury/inflammatory response is not understood. In this study, we demonstrate that AnxA1-deficient animals exhibit increased susceptibility to dextran sulfate sodium (DSS)-induced colitis with greater clinical morbidity and histopathologic mucosal injury. Furthermore, impaired recovery following withdrawal of DSS administration was observed in AnxA1 (-/-) animals compared with wild-type (WT) control mice that was independent of inflammatory cell infiltration. Since AnxA1 exerts its anti-inflammatory properties through stimulation of ALX/FPRL-1, we explored the role of this receptor-ligand interaction in regulating DSS-induced colitis. Interestingly, treatment with an ALX/FPRL-1 agonist, 15-epi-lipoxin A4 reversed the enhanced sensitivity of AnxA1 (-/-) mice to DSS colitis. In contrast, 15-epi-lipoxin A4 did not significantly improve the severity of disease in WT animals. Additionally, differential expression of ALX/FPLR-1 in control and DSS-treated WT and AnxA1-deficient animals suggested a potential role for AnxA1 in regulating ALX/FPRL-1 expression under pathophysiological conditions. Together, these results support a role of endogenous AnxA1 in the protective and reparative properties of the intestinal mucosal epithelium.

Role of the Protein C Pathway in the Extraintestinal Thrombosis Associated With Murine Colitis

Chronic inflammatory bowel diseases (IBD) are associated with an increased risk for thromboembolism. Although thrombosis is known to contribute to the morbidity and mortality of patients with IBD, the underlying mechanisms that contribute to the genesis of a hypercoagulable state during intestinal inflammation remain poorly defined. The objective of this study was to determine whether the protein C pathway contributes to the enhanced extraintestinal thrombosis that is associated with dextran sodium sulfate (DSS)-induced colitis in mice. Microvascular thrombosis was induced in cremaster muscle microvessels of normal and colitic mice using a light/dye injury model. DSS colitis enhanced thrombus formation in cremaster arterioles of wild-type mice. The DSS-induced thrombosis response was greatly attenuated in transgenic mice over expressing the endothelial protein C receptor. Activated protein C (APC), administered to colitic WT mice immediately prior to photoactivation, also afforded protection against thrombosis, and an anti-APC antibody enhanced thrombus formation. These findings indicate that elevated APC levels, derived from either endogenous or exogenous sources, confer protection against the extraintestinal thrombosis that accompanies colonic inflammation.

Suppressor of cytokine signaling-1 ameliorates dextran sulfate sodium-induced colitis in mice

Inflammatory bowel disease (IBD) is a chronic disorder of the gastrointestinal tract. Although the etiology and pathogenesis of IBD remain unknown, pro-inflammatory cytokines including IFN-gamma play an important role in the development of IBD. Suppressor of cytokine signaling-1 (SOCS-1) is a crucial inhibitor of cytokine signaling, particularly of IFN-gamma. In this study, we investigated the role of SOCS-1 in the development of murine dextran sulfate sodium (DSS)-induced colitis, a model of colitis resembling human IBD. SOCS-1 heterozygous (SOCS-1(+/-)) and wild-type (WT) mice were given 3% DSS dissolved in drinking water for 5 days. Activation and expression of signal transducers and activators of transcription (STAT) in colonic tissues were assessed by western blot analysis. The expression of CD4, IFN-gamma, IL-4, IL-17 and Forkhead box P3 (Foxp3) in colonic lamina propria lymphocytes was analyzed by flow cytometry and cytokine concentrations in serum were measured. DSS-treated SOCS-1(+/-) mice developed more severe colitis than DSS-treated WT mice. Enhanced activation of STAT1, a higher ratio of CD4(+)IFN-gamma(+) T cells and a lower frequency of Foxp3(+) regulatory T (Treg) cells, were observed in the colon of DSS-treated SOCS-1(+/-) mice compared with DSS-treated WT mice. DSS-treated SOCS-1(+/-) mice showed higher levels of IFN-gamma in sera than did DSS-treated WT mice. Furthermore, T cell-specific SOCS-1-conditional knockout mice developed more severe colitis than control mice after DSS administration. Our findings suggest that SOCS-1, particularly in T cells, prevents the development of DSS-induced colitis in mice by inhibiting IFN-gamma/STAT1 signaling and by subsequently regulating Treg cell development.