Cycles Of Dextran Sulfate Sodium Research Articles

Sodium orthovanadate protects against ulcerative colitis and associated liver damage in mice: insights into modulations of Nrf2/Keap1 and NF-κB pathways.

Ulcerative colitis (UC) is a prominent category of disease that is associated with bowel inflammation, it can occur at any period of life and is prevalently rising on a global scale. Dextran sulfate sodium (DSS) has been extensively used to develop colitis due to its ability to mimic human UC, providing consistent and reproducible inflammation, ulceration, and disruption of the epithelial barrier in the colon. Chronic inflammation in the gut can lead to alterations in the gut-liver axis, potentially impacting liver function over time, while direct evidence linking diversion colitis to liver damage is limited. Thus, the present study aims to assess the gut and liver damage against DSS and the possible molecular mechanisms. Forty-seven animals were randomly assigned to six groups. Ulcerative colitis was induced using 2.5% w/v DSS in three alternate cycles, each lasting 7 days, with 1-week remission periods in between. SOV (5 and 10 mg/kg, orally) and the standard drug 5-aminosalicylic acid (100 mg/kg, orally) were administered from the start of the 2nd DSS cycle until the end of the experiment. Biochemical parameters, ELISA, histopathological, and immunohistochemical analyses have been conducted to assess damage in the colon and liver. SOV significantly reduced colitis severity by lowering the DAI score, oxidative stress markers (LPS, IL-1β, MPO, nitrite), and restoring liver biomarkers (SGPT, SGOT). Histopathological findings supported these protective benefits in the liver and gut. Moreover, immunohistochemical analysis showed SOV enhanced the expression of the cytoprotective mediator Nrf2/Keap-1 and reduced the expression of inflammatory mediators NF-κB and IL-6. Present findings concluded that SOV demonstrated a dose-dependent effect against UC through anti-inflammatory and antioxidant pathways, with the highest dose of SOV 10 mg/kg having more significant (p < 0.001) results than the low dose of 5 mg/kg.

Low-dose dimethylfumarate attenuates colitis-associated cancer in mice through M2 macrophage polarization and blocking oxidative stress

Colitis-associated cancer (CAC) is an aggressive subtype of colorectal cancer that can develop in ulcerative colitis patients and is driven by chronic inflammation and oxidative stress. Current chemotherapy for CAC, based on 5-fluorouracil and oxalipltin, is not fully effective and displays severe side effects, prompting the search for alternative therapies. Dimethylfumarate (DMF), an activator of the nuclear factor erythroid 2–related factor 2 (NRF2), is a potent antioxidant and immunomodelatrory drug used in the treatment of multiple sclerosis and showed a strong anti-inflammatory effect on experimental colitis. Here, we investigated the chemotherapeutic effect of DMF on an experimental model of CAC. Male NMRI mice were given two subcutaneous injections of 1,2 Dimethylhydrazine (DMH), followed by three cycles of dextran sulfate sodium (DSS). Low-dose (DMF30) and high-dose of DMF (DMF100) or oxaliplatin (OXA) were administered from the 8th to 12th week of the experiment, and then the colon tissues were analysed histologically and biochemically. DMH/DSS induced dysplastic aberrant crypt foci (ACF), oxidative stress, and severe colonic inflammation, with a predominance of pro-inflammatory M1 macrophages. As OXA, DMF30 reduced ACF multiplicity and crypt dysplasia, but further restored redox status, and reduced colitis severity by shifting macrophages towards the anti-inflammatory M2 phenotype. Surprisingly, DMF100 exacerbated ACF multiplicity, oxidative stress, and colon inflammation, likely through NRF2 and p53 overexpression in colonic inflammatory cells. DMF had a dual effect on CAC. At low dose, DMF is chemotherapeutic and acts as an antioxidant and immunomodulator, whereas at high dose, DMF is pro-oxidant and exacerbates colitis-associated cancer.

Morphological Characteristics of Colon Tumors in Mice with Different Tolerance to Hypoxia.

In adult male C57BL/6 mice with high (HR) and low (LR) resistance to hypoxia, morphological features of colon tumors and blood parameters were evaluated 70 days after intraperitoneal injection of azoxymethane and subsequent consumption of 3 cycles of dextran sulfate sodium. On macroscopic analysis, tumors were found in the distal colon in 35% (7 of 20 animals) of HR and 31% (4 of 13 animals) of LR animals. Microscopic analysis of the distal colon revealed tumors in 75% (15 of 20 animals) of HR and 69% (9 of 13 animals) of LR mice. The tumors were presented by areas of glandular intraepithelial neoplasia and adenocarcinomas; the incidence and the area of the tumors did not differ in groups of HR and LR mice. The number of neuroendocrine and goblet cells in the distal colon mucosa in the areas of tumors was similar in the compared groups. However, in both HR and LR mice of the experimental groups, the content of goblet cells in tumors was lower and the content of endocrine cells was higher than in the corresponding control groups. In the peripheral blood, the erythrocyte count and hemoglobin content decreased in HR and LR mice of the experimental groups; the relative number of monocytes increased only in HR mice and the absolute number of lymphocytes and monocytes decreased in LR mice. Thus, 70 days after azoxymethane administration and dextran sulfate sodium consumption, the tumors in mice were presented by glandular intraepithelial neoplasia and adenocarcinomas, and their incidence and area did not differ between animals with different tolerance to hypoxia.

Abstract 295: Fibroblast-specific endoglin deletion alters colonic immune infiltrate in premalignant colorectal lesions

Abstract Background: Endoglin expression on cancer-associated fibroblasts has been reported to promote tumor growth and metastasis, especially in colorectal cancer (CRC). However, little research has been done on the function of endoglin-expressing fibroblasts in the early stages of CRC and how it interacts with the host immune system. Here, we investigated the role of endoglin expression on fibroblasts during intestinal inflammation and tumorigenesis in a chemically induced colitis-associated CRC model. Methods: We generated two inducible fibroblast-specific endoglin (ENG) knockout mice Collagen1a1 CreERT2.ENGfl/fl (ENGCol1a1-/−) and Collagen1a2-CreERT.ENGfl/fl (ENGCol1a2-/−). Cre-mediated recombination in mice was induced by oral administration of tamoxifen for three consecutive days. Polyp formation was induced by a single injection of azoxymethane (AOM), followed by three cycles of dextran sodium sulphate (DSS). Analysis of the immune cell composition in the colons and polyps was done using flow cytometry. Hematoxylin and eosin (H&amp;E) staining was performed to evaluate intestinal inflammation. Results: ENGCol1a1-/− mice showed a considerably higher number of colonic lesions than non-induced controls, whereas in the ENGCol1a2-/−, surprisingly no significant difference in the number of lesions was observed. Flow cytometry analysis revealed, compared to controls, the lesions of ENGCol1a1-/− mice had a significant increase in F4/80+ Ly6C− macrophages and Ly6G+ neutrophils, while those of ENGCol1a2-/− mice had a marked decrease in CD11C+ dendritic cells. Given the changes in immune cell infiltration, we also assessed acute inflammation, using a short-term DSS experiment. Interestingly, ENGCol1a1-/− mice lost significantly more weight compared to controls, indicating increased inflammation. Although weight loss did not differ in Col1a2 mice between the two groups, ENGCol1a2-/− mice had a much lower H&amp;E staining score, indicating less colonic damage and inflammation. Furthermore, a decrease in F4/80+ Ly6C− macrophages was observed in the colons of both ENGCol1a1-/- and ENGCol1a2-/− mice, despite no change in the overall CD45+ immune cell population. Notably, the number of Ly6G+ neutrophils was decreased in the colons of ENGCol1a1-/− mice but increased in ENGCol1a2-/− mice. In addition, a decrease in CD11C+ dendritic cells was seen in the colons ENGCol1a2-/− mice. Conclusion: Our results suggest that AOM/DSS-induced polyp formation and infiltrating immune cells differ in ENGCol1a1-/− and ENGCol1a2-/− mice. Fibroblast-specific deletion of endoglin can reduce macrophage and dendritic cell infiltration, colonic damage, and protect mice from DSS colitis. Citation Format: Subinuer Abudukelimu, Mark J. Schoonderwoerd, Madelon Paauwe, Eveline S. de Jonge-Muller, Stef G. Janson, Nadine van Montfoort, Lukas JAC Hawinkels. Fibroblast-specific endoglin deletion alters colonic immune infiltrate in premalignant colorectal lesions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 295.

Human umbilical cord/placenta mesenchymal stem cell conditioned medium attenuates intestinal fibrosis in vivo and in vitro

BackgroundA significant unmet need in inflammatory bowel disease is the lack of anti-fibrotic agents targeting intestinal fibrosis. This study aimed to investigate the anti-fibrogenic properties and mechanisms of the conditioned medium (CM) from human umbilical cord/placenta-derived mesenchymal stem cells (UC/PL-MSC-CM) in a murine intestinal fibrosis model and human primary intestinal myofibroblasts (HIMFs).MethodsUC/PL-MSC-CM was concentrated 15-fold using a 3 kDa cut-off filter. C57BL/6 mice aged 7 weeks old were randomly assigned to one of four groups: (1) control, (2) dextran sulfate sodium (DSS), (3) DSS + CM (late-phase treatment), and (4) DSS + CM (early-phase treatment). Chronic DSS colitis and intestinal fibrosis was induced by three cycles of DSS administration. One DSS cycle consisted of 7 days of oral DSS administration (1.75%, 2%, and 2.5% DSS), followed by 14 days of drinking water. UC/PL-MSC-CM was intraperitoneally administered in the late phase (from day 50, 10 times) or early phase (from day 29, 10 times) of DSS cycles. HIMFs were treated with TGF-β1 and co-treated with UC/PL-MSC-CM (10% of culture media) in the cellular model.ResultsIn the animal study, UC/PL-MSC-CM reduced submucosa/muscularis propria thickness and collagen deposition, which improved intestinal fibrosis in chronic DSS colitis. The UC/PL-MSC-CM significantly reduced the expressions of procollagen1A1 and α-smooth muscle actin, which DSS significantly elevated. The anti-fibrogenic effect was more apparent in the UC-MSC-CM or early-phase treatment model. The UC/PL-MSC-CM reduced procollagen1A1, fibronectin, and α-smooth muscle actin expression in HIMFs in the cellular model. The UC/PL-MSC-CM downregulated fibrogenesis by suppressing RhoA, MRTF-A, and SRF expression.ConclusionsHuman UC/PL-MSC-CM inhibits TGF-β1-induced fibrogenic activation in HIMFs by blocking the Rho/MRTF/SRF pathway and chronic DSS colitis-induced intestinal fibrosis. Thus, it may be regarded as a novel candidate for stem cell-based therapy of intestinal fibrosis.

Sex differences in colonic gene expression and fecal microbiota composition in a mouse model of obesity-associated colorectal cancer

This study investigated the sex-specific correlation between obesity and colorectal cancer emphasizing a more pronounced association in males. Estrogen, chromosomal genes, and gut bacteria were assessed in C57BL6/J male, female and ovariectomized (OVX) female mice, subjected to either a low-fat diet (LFD) or high-fat diet (HFD) for 14 weeks. Induction of colon tumor involved azoxymethane (10 mg/kg) administration, followed by three cycles of dextran sulfate sodium. Male mice on HFD exhibited higher final body weight and increased colon tumors compared to females. Colonic mucin 2 expression was significantly higher in females. HFD-modulated differentially expressed genes numbered 290 for males, 64 for females, and 137 for OVX females. Only one up-regulated gene (Gfra3) overlapped between females and OVX females, while two down-regulated genes (Thrsp and Gbp11) overlapped between males and OVX females. Genes up-regulated by HFD in males were linked to cytokine-cytokine interaction, HIF-1 signaling pathway, central carbon metabolism in cancer. Sex-specific changes in gut microbial composition in response to HFD were observed. These findings suggest a male-specific vulnerability to HFD-induced colon tumor formation, implicating key genes and colonic bacteria in colon tumorigenesis.

IRE1A-XBP1 CONTROLS GROUP 2 INNATE LYMPHOID CELLS IN INTESTINAL INFLAMMATION AND FIBROSIS

Abstract BACKGROUNDS Group 2 innate lymphoid cells (ILC2s) are increased in peripheral blood and intestinal samples from patients with ulcerative colitis or Crohn’s disease and murine models of IBD. ILC2s produce a large amount of type 2 cytokines including IL-5 and IL-13 and growth factor amphiregulin (AREG) that induce goblet cell hyperplasia and mitigate acute inflammation in the gut. On the other hand, prolonged or uncontrolled ILC2 activation may contribute to chronic inflammation and tissue fibrosis. However, the regulatory mechanisms and functional impact of ILC2s in IBD remains elusive. The IRE1a-XBP1 pathway orchestrates cellular stress response that plays a key role in intestinal inflammation. METHODS We generated conditional knockout Ire1aflo/floxIl5-Cre (Ire1aΔIl5) with Ire1a deleted in ILC2s. Rag-/-Ire1aΔIl5 mice were also generated to exclude possible contribution of T cells during chronic inflammation. Dextran sodium sulfate (DSS) was given in drinking water to induce acute/chronic colitis and intestinal fibrosis. We also collected colonic biopsies from healthy individuals and sort-purified ILC2s for flow cytometry analysis. RESULTS Mouse intestinal ILC2s express high level of Ire1a. The number of ILC2s increased in small intestine (SI) and colon of Ire1aΔIl5 mice compared to Il5-Cre controls. Additionally, SI and colonic Ire1aΔIl5 ILC2s produced more IL-5, IL-13, and AREG after ex vivo stimulation with IL-33 and IL-25. Upon DSS challenge, Ire1aΔIl5 mice exhibited milder signs of disease including weight loss, clinical scores as well as colon shortening and histology (Figure 1), which was accompanied by elevations in ILC2-derived cytokines and AREG as well as heightened goblet cell differentiation and mucin production. In response to 3 cycles of DSS, Rag-/-Ire1aΔIl5 mice developed worsened chronic colitis, fibrosis, and increased fibrosis markers including collagens and vimentin. Intraperitoneal injection of neutralizing antibody against IL-13 or AREG reversed the phenotype. In sort-purified human ILC2s, selective IRE1 inhibitor 4u8C enhanced production of IL-13, while selective IRE1 activator IXA4 suppressed IL-13 in response to IL-2, IL-7, and IL-33 ex vivo (Figure 2). CONCLUSIONS We demonstrate that IRE1a-XBP1 constrains cytokine production by intestinal ILC2s during inflammation. IRE1a deficiency in ILC2s protected against acute colitis with heightened type 2 cytokines and AREG; while loss of IRE1a exacerbated chronic colitis and fibrosis. We identified IRE1a as a potential therapeutic target for fibrostenotic CD. Figure 1 Ire1aΔIl5 mice harbor elevated ILC2s (A) and produce increased IL-5, IL-13, and AREG by (B) SI and (C) colonic ILC2s ex vivo. (D-H) Ire1aΔIl5 mice are protected from acute DSS colitis. *P&amp;lt;0.001. Figure 2 IRE1 modulators control IL-13 production by human ILC2s. **P&amp;lt;0.01.

Yogurt Prevents Colitis-Associated Colorectal Cancer in Mice.

Epidemiological studies indicate an inverse correlation between yogurt consumption and colorectal cancer (CRC), but whether there is a cause-and-effect relationship has not yet been validated. This study aims to investigate the effects and possible mechanisms of yogurt on colitis-associated colorectal cancer (CAC) in mice. Experimental CAC is induced by azoxymethane (AOM, 10mgkg-1 , ip) followed by three cycles of dextran sulfate sodium (DSS, 3%) treatment. Colitis is induced by adding DSS (3%) in drinking water for 5 days. Primary mouse macrophages are isolated for mechanistic studies. Data clearly show that yogurt (15gkg-1 body weight) significantly reduces the multiplicity of colonic neoplasms by 38.83% in mice. Yogurt protects mice from colitis dependent on lactate receptor GPR81. The deficiency of Gpr81 exacerbates colitis and CAC in mice. Further investigation reveals that GPR81 may be dispensable for gut barrier function but essential for colonic mucosal repair. d-lactate in yogurt can activate GPR81 to suppress proinflammatory macrophage polarization, thereby facilitating inflammatory resolution after colonic injury and consequently suppressing CAC progression. Yogurt effectively protects against colitis-associated colorectal tumorigenesis in mice, and this study provides a rationale for introducing yogurt supplementation to patients with chronic inflammatory bowel diseases.

P096 Characterization of suppressive immune cell subsets in mouse models of colitis-associated colorectal cancer

Abstract Background Chronic colonic inflammation in inflammatory bowel disease (IBD) patients increases the risk of colitis-associated cancer (CAC). Cancer development in CAC is different from that observed in sporadic colorectal cancer (CRC). Most studies have focused on the role of pro-inflammatory immune cell subsets in CAC, yet it is becoming increasingly clear that immunosuppression may also drive cancer progression. We hypothesize that emergence of immunosuppressive cell subsets as a result of chronic intestinal inflammation, dampens anti-tumor immune responses and accelerates the development of CAC. Methods To simulate cancer formation in the background of chronic inflammation, mice were treated with azoxymethane (AOM) followed by three cycles of dextran sodium sulphate (DSS). Apc Min/+ mice treated with AOM were used to model sporadic CRC. After 10 weeks, immune cells from the proximal and distal colonic lamina propria, mesenteric lymph nodes (MLNs) and tumors were obtained for microscopy analyses and processed to obtain single cell suspensions for flow cytometry. Immune cell populations were studied by conventional flow cytometry and immunofluorescence. Results We observed a significant increase in absolute numbers of PD1+CTLA4+TIM3+ ‘exhausted’ CD4+ T cells in the lamina propria from proximal colons in CAC mice as compared to sporadic CRC mice. Interestingly, these numbers showed a positive correlation with the inflammation score in AOM/DSS, demonstrating that chronic inflammation is associated with the emergence of ‘exhausted’ T cells. In addition, we observed a trend to higher numbers of M2-like (MHCII-CD206+) suppressive macrophages and CD4+FoxP3+ regulatory T cells (Tregs) in tumors from CAC mice compared to sporadic CRC. When analysing T cell immune responses in the MLNs, we found a significant decrease in the ratio IFNγ/IL10-producing CD8+ T cells in CAC mice as compared to CRC mice, suggesting a shift towards an anti-inflammatory T cell response in CAC. Conclusion In summary, suppressive immune cell subsets were increased in colonic mucosa and tumors from CAC mice as a result of chronic inflammation, which may be associated with the development of dysplasia. Further functional studies are necessary to prove the role of suppressive immune subsets in IBD-associated dysplasia- and carcinoma progression.

NR4A1 modulates intestinal smooth muscle cell phenotype and dampens inflammation-associated intestinal remodeling.

Stricture formation is a common complication of Crohn's disease (CD), driven by enhanced deposition of extracellular matrix (ECM) and expansion of the intestinal smooth muscle layers. Nuclear receptor subfamily 4 group A member 1 (NR4A1) is an orphan nuclear receptor that exhibits anti-proliferative effects in smooth muscle cells (SMCs). We hypothesized that NR4A1 regulates intestinal SMC proliferation and muscle thickening in the context of inflammation. Intestinal SMCs isolated from Nr4a1+/+ and Nr4a1-/- littermates were subjected to shotgun proteomic analysis, proliferation, and bioenergetic assays. Proliferation was assessed in the presence and absence of NR4A1 agonists, cytosporone-B (Csn-B) and 6-mercaptopurine (6-MP). In vivo, we compared colonic smooth muscle thickening in Nr4a1+/+ and Nr4a1-/- mice using the chronic dextran sulfate sodium (DSS) model of colitis. Second, SAMP1/YitFc mice (a model of spontaneous ileitis) were treated with Csn-B and small intestinal smooth muscle thickening was assessed. SMCs isolated from Nr4a1-/- mice exhibited increased abundance of proteins related to cell proliferation, metabolism, and ECM production, whereas Nr4a1+/+ SMCs highly expressed proteins related to the regulation of the actin cytoskeleton and contractile processes. SMCs isolated from Nr4a1-/- mice exhibited increased proliferation and alterations in cellular metabolism, whereas activation of NR4A1 attenuated proliferation. In vivo, Nr4a1-/- mice exhibited increased colonic smooth muscle thickness following repeated cycles of DSS. Activating NR4A1 with Csn-B, in the context of established inflammation, reduced ileal smooth muscle thickening in SAMP1/YitFc mice. Targeting NR4A1 may provide a novel approach to regulate intestinal SMC phenotype, limiting excessive proliferation that contributes to stricture development in CD.

Therapeutic Potential of the Combination of Pentoxifylline and Vitamin-E in Inflammatory Bowel Disease: Inhibition of Intestinal Fibrosis.

Background: Although intestinal fibrosis is a consequence of recurrent inflammation in Inflammatory bowel disease (IBD), alleviating inflammation alone does not prevent the progression of fibrosis, suggesting that the development of direct anti-fibrotic agents is necessary. This study aimed to evaluate the anti-fibrotic properties of combination treatment with pentoxifylline (PTX) and vitamin E (Vit-E) on human primary intestinal myofibroblasts (HIMFs) and the therapeutic potential of the combination therapy in murine models of IBD. Methods: HIMFs were pretreated with PTX, Vit-E, or both, and incubated with TGF-β1. We performed Western blot, qPCR, collagen staining, and immunofluorescence to estimate the anti-fibrotic effects of PTX and Vit-E. The cytotoxicity of these was investigated through MTT assay. To induce murine models of IBD for in vivo study, C57BL/6 mice were treated with repeated cycles of dextran sulfate sodium (DSS), developing chronic colitis. We examined whether the combined PTX and Vit-E treatment would effectively ameliorate colonic fibrosis in vivo. Results: We found that the co-treatment with PTX and Vit-E suppressed TGF-β1-induced expression of fibrogenic markers, with decreased expression of pERK, pSmad2, and pJNK, more than either treatment alone in HIMFs. Neither PTX nor Vit-E showed any significant cytotoxicity in given concentrations. Consistently with the in vitro results, the co-administration with PTX and Vit-E effectively attenuated colonic fibrosis with recovery from thickening and shortening of colon in murine models of IBD. Conclusions: These findings demonstrated that the combination of PTX and Vit-E exhibits significant anti-fibrotic effects in both HIMFs and in vivo IBD models, providing a promising therapy for IBD.

Abstract 2539: Macrophage derived WNTs regulate immune checkpoint expression and inflammation induced colon cancer

Abstract Colorectal cancer is the third most common cancer in the world. Patients with inflammatory bowel disease are at an elevated risk for developing colon cancer. Macrophages are important members of tumor microenvironment (TME) and promote inhibition of anti-tumor immunity by activation of immune checkpoints and other neo-antigens expressions. However, paracrine signals regulating these checkpoint expressions in macrophages need to be determined. Macrophage-derived WNT ligands play important role in tissue homeostasis in multiple organs. In the present study we have demonstrated that inhibition of macrophage-derived WNT promotes inflammation-induced colon cancer. In both the mice model and human in vitro model we have shown that the absence of macrophage-derived WNT promotes immunosuppressive microenvironment with upregulation of immune checkpoint expressions in tumor-associated macrophages and T cells. Release of WNT ligands expressed in macrophages was inhibited by using Csf1r.iCre; Porcn fl/fl mice where gene expressing PORCUPINE involved in post translational modification and release of WNT ligands was deleted in macrophages using Cre recombinase expressed under Csf1r promoter. In these mice and wild type littermates, Inflammation-induced colon cancer was developed using AOM-DSS treatment. Mice were treated with a single dose of 16mg/kg of body weight Azoxymethane (AOM) (i.p.) and subsequent three cycles of 3.5% of Dextran sodium sulfate (DSS) (supplementation in drinking water). Development of colon cancer was monitored using small animal colonoscopy. After 3 cycles of DSS Csf1r.iCre; Porcn fl/fl mice demonstrated a significantly higher number of colon tumors compared to wild type littermate (Macro size: p&amp;lt;0.0005, Micro size: p&amp;lt;0.013). Moreover, flow cytometric analysis of tumor tissue demonstrated that in Csf1r.iCre; Porcn fl/fl mice both PD1 and PDL1 expression was high in tumor-associated macrophages compared to wild type mice. PDL1 expression was also high in colon tumor tissue, myeloid-derived suppressor cells (MDSCs) and helper T cells in Csf1r.iCre; Porcn fl/fl mice. Similar observation was made in ex-vivo co-culture model using human pan T cells, HTB-38 human colon cancer cells and human peripheral blood mononuclear cell (PBMC) derived macrophages pre-treated with PORCUPINE inhibitor C59. Therefore, our present study demonstrated for the first time that macrophage-derived WNT is required to modulate the immunosuppressive role of macrophages in inflammation-induced colon cancer. Inhibition of WNT release specific from macrophages promotes PDL1 expression in colon cancer cells and other cell types in TME including MDSCs, and T helper cells. Further studies are ongoing to determine the mechanism of autocrine action of WNT ligands in tumor-associated macrophages to maintain anti-tumor immunity. Citation Format: Subhrajit Saha, Pooja Gupta, Rishi Man Chugh, Ximena Diaz Olea, Tanu Arora, Payel Bhanja. Macrophage derived WNTs regulate immune checkpoint expression and inflammation induced colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2539.

Abstract 4218: 6-gingerol upregulated CD8+ T cells function in mice model of colorectal cancer via modulation of programmed cell death protein 1/programmed death-ligand1 and cytotoxic T-lymphocyte-associated protein 4 receptor signaling

Abstract Colorectal cancer (CRC) is the third leading cause of cancer-related death in the United States, with about 149,500 new cases of CRC diagnosed in 2021. Although various treatments are available for CRC, survival rates of CRC patients remain very poor. In the last few decades, Inhibition of immune checkpoint proteins have been shown to be a promising strategy for CRC treatments, thus allowing T cells to destroy CRC cells. 6-gingerol, an active compound present in fresh ginger have been shown to possess anti-cancer and immunomodulatory properties. We have previously described the anti-metastatic effect 6-gingerol in CRC. However, there is paucity of information on the effect 6-gingerol on CD8+ T cell function and immune checkpoints proteins in CRC. Therefore, this study investigated the effect of 6-gingerol on CD8+ T cells function and immune check point proteins in using mice model of CRC and system biology approach. Male BALB/c mice were divided into four groups of 10 mice each. Group 1 mice serve as control, group 2 mice received 6-gingerol 10mg/kg/day by oral gavage for 10 weeks. Group 3 (CRC model) mice received a single dose of azoxymethane 10mg/kg (Intraperitoneal) and on the seventh day they received three cycles of dextran sulfate sodium (DSS) 2% (W/V) in drinking water (each cycle is defined as one week of 2% DSS followed by two weeks of plain drinking water) Group 4 mice received 6-gingerol 10mg/kg/day by oral gavage in combination with single dose of azoxymethane 10mg/kg (iP) and three cycles of 2% DSS (W/V) in drinking water. Administration of 6-gingerol to AOM/DSS treated mice increase the percentage (%) gated cells population of CD8+Tcells and cytotoxic T-lymphocyte-associated protein 4 (CTLA4) with concomitant decrease in % gated population of Programmed cell death protein 1 (PD-1) and B-7 proteins as compared to AOM/DSS only treated mice. Furthermore, there was decrease expression of PD-L1 on colon tissues of 6-gingerol treated mice when compared with AOM/DSS treated mice colon. Also, using system biology analysis, we observed a high binding energy of 6-gingerol with PD-1/PD-L1 Complex, PD-L1, Dimeric PD-L1 and CTLA-4/B7-2 complex proteins. The result obtained from this study showed that 6-gingerol inhibited immune check point proteins and upregulated CD8+ T cells function in colorectal cancer via modulation of PD-1/PD-L1 and CTLA4 receptor signaling, and as such 6-gingerol could be a promising natural product for CRC immunotherapy. Citation Format: Babajide O. Ajayi, Anuoluwapo Adeshina. 6-gingerol upregulated CD8+ T cells function in mice model of colorectal cancer via modulation of programmed cell death protein 1/programmed death-ligand1 and cytotoxic T-lymphocyte-associated protein 4 receptor signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4218.

A144 CHRONIC DSS LEADS TO ALTERED EXPRESSION OF IRAK4/TPL2 PATHWAYS IN UC-COLONIZED MICE

BackgroundUlcerative colitis (UC) is one of two forms of inflammatory bowel disease (IBD). The exact cause of IBD is unknown but altered host-microbe interactions and genetic susceptibility are involved in its pathogenesis. Many patients with IBD do not respond to biological therapies targeting single cytokines, therefore new therapies that target common immune pathways are being developed and need to be tested in relevant preclinical models. Previously we have shown that mice colonized with UC microbiota upregulated genes related to inflammation without induction of colitis compared to healthy volunteer-colonized mice. We thus investigated whether IRAK4 and TPL2-induced pathways, new therapeutic targets in development upstream of inflammatory cytokine gene activation, are upregulated in mice colonized with UC microbiota and chronic colitis.AimsOur aim was to characterize TPL2 and IRAK4 signalling pathways and T cell phenotypes in UC-colonized mice following chronic low-dose dextran sodium sulfate (DSS) colitis.Methods10-16-week-old germ-free C57BL/6 mice were colonized with fecal microbiota from a patient with UC experiencing a flare (n=16). Mice were housed in a gnotobiotic facility during the experiment. Three weeks following colonization, colitis was induced in half of the mice by three cycles (5 d each; 2.0%, 1.5% and 1.5%, respectively) of DSS in drinking water with a 5-d wash-out period between cycles. All mice were fed a control diet (7004, Teklad). Fecal samples were collected weekly. At sacrifice, disease activity (colon length, occult blood in feces, stool consistency, and spleen weight) was measured and colon tissue was collected for histological analysis and RNA sequencing. Mesenteric lymph nodes (MLNs) were acquired for flow cytometry to analyze T cell phenotypes.ResultsIn UC-colonized mice, chronic low-dose DSS induced softer feces (p<0.0001), shorter colon length (p<0.0001), and increased spleen weight (p<0.0001), compared with water treated mice. This was paralleled by detectable blood in stool, development of moderate colitis (DSS: 1.9+/-1.4 vs H2O: 0.5+/-0.2; p<0.02) and higher proportion of IL-17 (p=0.003) and IFN-γ-producing T cells (p=0.06) in MLNs compared with water treated mice. RNA sequencing revealed that inflammatory genes, mainly related to the IRAK4/TPL2 pathway (e.g., Gadd45b, Socs3, Il1b), were significantly increased (p≤0.05) in the colon of mice treated with DSS compared with water.ConclusionsWhen challenged with a chronic low-dose chemical injury agent, mice colonized with UC microbiota develop clinical and histological signs of colitis and upregulation of genes involved in inflammation like Gadd45b, Socs3, and Il1b. Thus, this model represents a new valuable tool for preclinical testing of new drug candidates, such as those related to the modulation of IRAK4/TPL2 pathways.Funding AgenciesCCC

CHRONIC COLITIS ALTERS BRAIN ACTIVITY BY INDUCING HMGB1-MEDIATED PYROPTOSIS IN MICE

Abstract BACKGROUND AND AIM Chronic gut inflammation such as inflammatory bowel diseases is thought as being associated with neurodegenerative diseases in humans. The direct evidence for and the underlying mechanism of this brain-gut interaction, however, remain elusive. METHODS We used manganese-enhanced magnetic resonance imaging (MEMRI) to assess brain functional activity from awake and freely moving mice. As a mouse model of chronic colitis, mice were treated with three cycles of dextran sulfate sodium. We performed the passive avoidance test, which is a fear-motivated test that assesses short-term and long-term memory. Mouse hippocampus tissues and microglial cells were subjected to GC-MS, immunofluorescence staining, ELISA, and immunoblotting analysis to examine how the inflamed gut is linked to the brain pathology. RESULTS We found that manganese ion uptake, indicative of Ca2+ influx into neuronal cells, and accumulation are dramatically reduced in the hippocampus of chronic colitis mice compared to control mice. Long-term memory is declined in chronic colitis mice. Neuroinflammatory signals, including IL-1β production and the activation of Caspase-1, Caspase-11, and Gasdermin (GSDM), are induced in the hippocampus of chronic colitis mice. High-mobility group box 1 (HMGB1) level is elevated both in the serum and in the hippocampus of chronic colitis mice; however, lipopolysaccharide (LPS) levels remain at low levels without significant changes in these samples. The blood-brain barrier permeability is increased in chronic colitis mice. In the presence of LPS, accordingly, HMGB1 treatment induces the activation of Caspase-11 and GSDM in mouse microglial cell line SIM-A9. CONCLUSION Our findings suggest that HMGB1 released from the inflamed intestine may move to the brain through the blood circulatory system; in conjunction with a low level of endogenous LPS, elevated HMGB1 can subsequently activate Caspase-mediated inflammatory responses in the brain. This study implies that chronic gut inflammation may alter brain activity in mice.

P073 Eosinophil depletion partially protects from colonic inflammation, but increases colonic collagen deposition in a DSS colitis model

Abstract Background The role of eosinophils in intestinal inflammation and fibrosis in inflammatory bowel disease (IBD) is largely unknown. Therefore, we assessed the functional role of eosinophils in a chronic murine model of colitis and associated fibrosis via anti-CCR3 mediated eosinophil depletion. Methods 6-8-week-old C57BL/6 RAG-/- mice received three cycles of dextran sodium sulphate (DSS) (1.75% - 2.25% - 2.25%) each interspersed with 14 days of recovery. Twice weekly, anti-CCR3 antibody (n=8), isotype (n=8) or saline injections (n=8) were given intraperitoneally. At the same timepoints, the disease activity index (DAI; mouse weight, stool consistency and presence of blood) was determined. At sacrifice, colonic damage was scored macroscopically (presence of hyperaemia, adhesions and length and degree of colon affected by inflammation). Colonic single cells were isolated and stained for flow cytometry, where eosinophils were characterized as CD45+ CD11b+ Siglec-F+ CD117- cells. Intestinal fibrosis was scored via colon weight/length, collagen deposition (using a colorimetric hydroxyproline assay) and COL1A1 expression by PCR. Results Anti-CCR3 mediated eosinophil depletion resulted in decreased disease activity compared to the other DSS treated groups injected with saline or isotype, determined by the area under the curve of the DAI (74.6±18.4 vs. 127.5±42.9 and 136.9±33.6, p=0.01 and p=0.0008 respectively) (figure 1). The macroscopic damage score also suggested eosinophil depleted mice to be partially protected from colonic inflammation compared to the saline and isotype injected mice that received DSS (1.1±1.0 vs. 2.1±1.2 and 3.0±0.7, p=0.09 and p=0.001 respectively) (figure 1). Colon weight/length and hydroxyproline assay showed a trend towards increased fibrosis in the anti-CCR3 injected group compared to saline (p=0.03 and 0.07, respectively) but not isotype (p=0.3 and 0.1, respectively) injected groups (figure 2). However, COL1A1 expression levels were significantly increased in the eosinophil depleted mice compared to the saline and isotype injected mice receiving DSS (43.2±11.4 vs. 23.3±8.7 and 30.1±11.0, p=0.002 and 0.04 respectively) (figure 2), indicating increased collagen deposition. Conclusion Eosinophil depletion via intraperitoneal anti-CCR3 injections resulted in partial protection against colonic inflammation, but was associated with increased collagen production. Caution is therefore needed when designing therapeutic interventions targeting eosinophils.

Abstract 2842: 6-gingerol attenuates metastasis in aged mice model of colorectal cancer through modulation of epithelial-mesenchymal transition mediators genes and e-cadherin upregulation

Abstract Colorectal cancer (CRC) is the third most common cancer in America, and the third cause of cancer related death. Metastasis is the major cause of death in patient with CRC. Many CRC are likely to spread from primary site to the other parts of the body before and after detection of malignant tumor. 6-gingerol, the bioactive component of fresh ginger have been reported to have myriad of biological activities including anti-inflammatory and antioxidant properties. We have previously reported the anti-CRC effect of 6-gingerol. However there is dearth of information on the anti-metastatic effect 6-gingerol in CRC. Thus, the effect of 6-gingerol on epithelial-mesenchymal transition mediators' genes and E-Cadherin repression in aged mice model of CRC was assessed in this study. Following institutional ethical approval. Aged male BALB/c mice (10 months) were divided into four groups of 10 mice each. Mice were fed with AIN-93 diet and allowed access to water ad libitum. Group 1 mice serve as control, group 2 mice received 6-gingerol 10mg/kg/day by oral gavage for 10 weeks. Group 3 mice received a single dose of azoxymethane (AOM) 10mg/kg (iP) and on the seventh day they received three cycles of dextran sulfate sodium (DSS) 2% (W/V) in drinking water ( each cycle is define as one week of DSS followed by two weeks of plain drinking water) Group 4 mice received 6-gingerol 10mg/kg/day by oral gavage in combination with single dose of azoxymethane 10mg/kg (iP) and on the seventh day they received three cycles of dextran sulfate sodium salt 2% (W/V) in drinking water. Mice were humanely sacrificed under anesthesia and colon tissues were processed for RT-PCR and multiplex immunohistochemistry analysis. Mice that received both 6-gingerol and AOM and DSS showed a significant decreased expression of Lymphocyte function-associated antigen-1, N-cadherin, SNAIL 1, ZEB 1, AP4 and HMGA1 with concomitant Upregulation miR-34, miR-15a, miR-218 and miR-302 expression as compared with the AOM and DSS only group. The result obtained from this study showed 6-gingerol inhibited metastasis through modulation of epithelial-mesenchymal transition mediators' genes and E-Cadherin upregulation, and as such 6-gingerol could be a promising anti-metastatic agent in CRC treatment. Citation Format: Babajide Oluwaseun Ajayi, Ayomiposi Adenigba, Anuoluwapo Adeshina. 6-gingerol attenuates metastasis in aged mice model of colorectal cancer through modulation of epithelial-mesenchymal transition mediators genes and e-cadherin upregulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2842.

Moringa oleifera leaves alleviated inflammation through downregulation of IL-2, IL-6, and TNF-α in a colitis-associated colorectal cancer model

New chemopreventive alternatives are needed due to the rising worldwide incidence of colorectal cancer. The objective was to evaluate the chemopreventive activity of Moringa oleifera leaves (MO) in a colitis-associated colon carcinogenesis model. We hypothesized that MO contain bioactive compounds capable of modulating the expression of genes involved in the inflammatory response and carcinogenesis. Forty-eight male mice (CD-1) were divided into six groups; 1: Healthy control; 2: Positive control induced with azoxymethane (AOM, 10 mg/Kg body weight, intraperitoneal injection) and three cycles of dextran sodium sulfate (DSS, 1.5% in drinking water); groups 3, 4, and 5 were induced with AOM/DSS and supplemented with 5%, 10%, and 20% of MO, respectively; group 6: had no disease induction and supplemented with 20% of MO. Mice were treated for 12 weeks and euthanized. Significant differences (p < 0.05) were found for the moringa-administered groups in morphological and histopathological parameters compared to the AOM/DSS control. A decrease in myeloperoxidase activity (~50%) and lipid peroxidation (1.9–3.1 times) were found in groups with 10% and 20% of MO compared to the AOM/DSS control (p < 0.05). The group supplemented with 10% MO showed a significant increase (~3 times) in butyrate and propionate in fecal and cecal content. Groups supplemented with 10%, and 20% MO showed a reduction in proinflammatory cytokines in serum (MCP-1, IL-6, TNF-α) compared to the AOM/DSS control. Treatment with 10% MO induced differential expression of 65 genes in colon tissue such as IL-2, IL-6, TNF, IL-1ß, and INF-γ. MO downregulated proinflammatory mediators showing chemopreventive properties against inflammatory response and colon carcinogenesis.

B. adolescentis ameliorates chronic colitis by regulating Treg/Th2 response and gut microbiota remodeling

ABSTRACT Inflammatory bowel disease (IBD) is defined as an immune dysregulation disease with poor prognosis. Various therapies based on gut microbe modulation have been proposed. In this study, we aim to explore the therapeutic effect of B. adolescentis on IBD, as well as the immune and microecology mechanism of B. adolescentis in IBD. The fecal level of B. adolescentis was decreased in the IBD patients compared with the normal people in our cohort and the GMrepo database. To further clarify the role of B. adolescentis in IBD, we induced chronic colitis with three cycles of dextran sulfate sodium (DSS). We found B. adolescentis gavage exhibited protective effects as evidenced by the significantly decreased diarrhea score, spleen weight, and increased colon length. Accordingly, the cumulative histological grading was decreased in the B. adolescentis administration group. In addition, tight junction protein and mucin family were enhanced after B. adolescentis treatment. Furthermore, distinct effects were found with decreased pro-inflammatory cytokines such as TNF-α, IL-6, IL-1β, IL-18, IL-22, IL-9 and increased anti-inflammatory cytokines IL-10, IL-4, IL-5. Importantly, the colon lamina propria in the B. adolescentis group consisted of more Treg and Th2 cells, which inhibited extreme gut inflammation. Additionally, 16srRNA sequencing showed an evident increase in the B:F ratio in the B. adolescentis group. In particular, B. adolescentis application inhibited the excessive growth of Akkermansia and Escherichia-Shigella in genus level. In conclusion, B. adolescentis refined the DSS-induced chronic colitis by stimulating protective Treg/Th2 response and gut microbiota remodeling. B. adolescentis regularly treatment might improve the therapeutic effects for inflammatory bowel disease.

Exercise Preconditioning Attenuates the Response to Experimental Colitis and Modifies Composition of Gut Microbiota in Wild-Type Mice.

This study investigated the suppressive effect of exercise preconditioning against colitis induced by high-fat diet (HF) plus dextran sulfate sodium (DSS) in wild-type mice. Male mice (C57BL/6) aged 6 weeks were assigned to standard chow (SC, n = 10) or HF (n = 10) or HF followed by DSS (HF+DSS, n = 10) or exercise preconditioning (EX) followed by HF+DSS (EX+HF+DSS, n = 10) for a total of 15 weeks. After 12 weeks of dietary treatments and/or exercise preconditioning, mice in the DSS groups were subjected to administration of 2 cycles of 5-day DSS (2% w/v) with a 7-day interval between cycles. HF resulted in colitis symptoms and histological changes, infiltration of immunity cells, decreased gut barrier proteins, increased pro-inflammatory and chemotactic cytokines and decreased anti-inflammatory cytokine such as adiponectin, which deteriorated after administration of DSS. Exercise preconditioning alleviated HF+DSS-induced colitis and caused significant modifications in gut microbiota: decreased Bacteroides vulgatus (p = 0.050) and increased Akkermansia muciniphila (p = 0.050). The current findings suggest that exercise preconditioning attenuates the severity of HF+DSS-induced colitis in C57BL/6 mice.