Defining the microbial transcriptional response to colitis through integrated host and microbiome profiling

The gut microbiome is significantly altered in inflammatory bowel diseases, but the basis of these changes is not well understood. We have combined metagenomic and metatranscriptomic profiling of the gut microbiome to assess modifications to both bacterial community structure and transcriptional activity in a mouse model of colitis. By using transcriptomic analysis of colonic tissue and luminal RNA derived from the host, we have also characterised how host transcription relates to the microbial transcriptional response in inflammation. In colitis, increased abundance and transcription of diverse microbial gene families involved in responses to nutrient deprivation, antimicrobial peptide production and oxidative stress support an adaptation of multiple commensal genera to withstand a diverse set of environmental stressors in the inflammatory environment. These data are supported by a transcriptional signature of activated macrophages and granulocytes in the gut lumen during colitis, a signature that includes the transcription of the key antimicrobial genes S100a8 and S100a9 (calprotectin). Genes involved in microbial resistance to oxidative stress, including Dps/ferritin, Fe-dependent peroxidase and glutathione S-transferase were identified as changing to a greater extent at the level of transcription than would be predicted by DNA abundance changes, implicating a role for increased oxygen tension and/or host-derived reactive oxygen species in driving transcriptional changes in commensal microbes

L-Threonine Supplementation During Colitis Onset Delays Disease Recovery

Dietary nutrients have emerged as potential therapeutic adjuncts for inflammatory bowel disease (IBD) given their impact on intestinal homeostasis through the modulation of immune response, gut microbiota composition and epithelial barrier stability. Several nutrients have already been associated with a protective phenotype. Yet, there is a lack of knowledge toward the most promising ones as well as the most adequate phase of action. To unveil the most prominent therapy candidates we characterized the colon metabolic profile during colitis development. We have observed a twofold decrease in threonine levels in mice subjected to DSS-induced colitis. We then assessed the effect of threonine supplementation in the beginning of the inflammatory process (DSS + Thr) or when inflammation is already established (DSS + Thr D8). Colitis progression was similar between the treated groups and control colitic mice, yet threonine had a surprisingly detrimental effect when administered in the beginning of the disease, with mice displaying a delayed recovery when compared to control mice and mice supplemented with threonine after day 8. Although no major changes were found in their metabolic profile, DSS + Thr mice displayed altered expression in mucin-encoding genes, as well as in goblet cell counts, unveiling an impaired ability to produce mucus. Moreover, IL-22 secretion was decreased in DSS + Thr mice when compared to DSS + Thr D8 mice. Overall, these results suggest that supplementation with threonine during colitis induction impact goblet cell number and delays the recovery period. This reinforces the importance of a deeper understanding regarding threonine supplementation in IBD.Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER) (NORTE-01-0145-FEDER-000013) and the Fundação para a Ciência e Tecnologia (FCT) (contracts PD/BD/106053/2015 to JG via Inter-University Doctoral Programme in Ageing and Chronic Disease – PhDOC, IF/00021/2014 to RS, IF/01390/2014 to ET, IF/01147/2013 to RD-O, IF/00735/2014 to AgC, SFRH/BPD/96176/2013 to CC, and SFRH/BPD/111100/2015 to LG). MS is a FCT investigator. The NMR data was acquired at CERMAX (Centro de Ressonância Magnética António Xavier) which is a member of the National NMR network with the support of Project LISBOA-01-0145-FEDER-007660info:eu-repo/semantics/publishedVersio

Suppressor of cytokine signaling 3 (SOCS3) is not an independent biomarker of colorectal adenoma risk

<p>Abstract</p> <p>Background</p> <p>Inflammation and its associated pathologies are increasingly suggested as risk factors for colorectal cancer (CRC) development. Previous research from our group has shown that increased levels of circulating, pro-inflammatory cytokines IL-6 and TNFα promote colorectal adenoma risk. Emerging data in mice and humans suggest that Suppressor of Cytokine Signaling 3 (SOCS3) may act as a tumor suppressor in the intestine, and decreased SOCS3 expression may promote CRC. As SOCS3 has been shown to inhibit the actions of IL-6 and TNFα in the intestine, we hypothesized that decreased SOCS3 expression in normal mucosa may predispose to adenomas and thus increase risk for CRC.</p> <p>Findings</p> <p>We examined SOCS3 mRNA levels in normal mucosa biopsies of 322 screening colonoscopy patients (93 with adenoma and 229 without adenoma) using real-time qRT-PCR. Logistic regression analysis was used to generate odds ratios (OR) and 95% confidence intervals to determine if low SOCS3 expression was associated with adenoma status. Median SOCS3 values did not differ between patients with or without adenoma. Logistic regression analysis showed no association (unadjusted or adjusted for age and sex) between SOCS3 and colorectal adenomas.</p> <p>Conclusions</p> <p>Low SOCS3 mRNA expression is not an independent biomarker of colorectal adenoma risk in the normal mucosa. SOCS3 silencing likely occurs later in CRC progression.</p

STAT3 links IL-22 signaling in intestinal epithelial cells to mucosal wound healing

Signal transducer and activator of transcription (STAT) 3 is a pleiotropic transcription factor with important functions in cytokine signaling in a variety of tissues. However, the role of STAT3 in the intestinal epithelium is not well understood. We demonstrate that development of colonic inflammation is associated with the induction of STAT3 activity in intestinal epithelial cells (IECs). Studies in genetically engineered mice showed that epithelial STAT3 activation in dextran sodium sulfate colitis is dependent on interleukin (IL)-22 rather than IL-6. IL-22 was secreted by colonic CD11c+ cells in response to Toll-like receptor stimulation. Conditional knockout mice with an IEC-specific deletion of STAT3 activity were highly susceptible to experimental colitis, indicating that epithelial STAT3 regulates gut homeostasis. STAT3IEC-KO mice, upon induction of colitis, showed a striking defect of epithelial restitution. Gene chip analysis indicated that STAT3 regulates the cellular stress response, apoptosis, and pathways associated with wound healing in IECs. Consistently, both IL-22 and epithelial STAT3 were found to be important in wound-healing experiments in vivo. In summary, our data suggest that intestinal epithelial STAT3 activation regulates immune homeostasis in the gut by promoting IL-22–dependent mucosal wound healing

Sex impacts Th1 cells, Tregs, and DCs in both intestinal and systemic immunity in a mouse strain and location-dependent manner

Background: Males and females have a different predisposition for the development of intestinal disorders, like inflammatory bowel disease (IBD). We hypothesized that sex specific differences in intestinal immune responses may underlie this bias. To test this hypothesis, we studied sex differences in immune cell populations in the Peyer's patches (PP). For comparison with systemic immunity, we studied spleen cells. Methods: Two mouse strains with different susceptibility for developing colitis (BALB/c and C57Bl/6) were used. Using flow cytometry, we measured the percentage of T cells, Th1, Th17, and Treg cells in the PP and spleen. In addition, we measured the percentages of NK cells, macrophages, myeloid, and lymphoid dendritic cells (DCs) and their expression of CD80 and CD103. Moreover, we measured percentages of monocyte subsets in the peripheral circulation. Results were tested using two-way ANOVA, p <0.05. Results: Males had a lower percentage of T cells in both PP and spleen (PP BALB/c 22.1 %, B6 13.6 %; spleen BALB/c 4.7 %, B6 19.9 %) but a higher percentage of Th1 cell in both tissues (PP BALB/c 350 %, B6 109.5 %; spleen BALB/c 48.7 %, B6 41.9 %) than females. They also had a higher percentage of Tregs in the spleen than females (BALB/c 20.5 %, B6 4.5 %). Furthermore, males had a higher percentage of CD80(+) DCs in both the PP and spleen (lymphoid DCs in PP BALB/c 104.7 %, B6 29.6 %; spleen BALB/c 72.2 %, B6 44.2 %; myeloid DCs in PP BALB/c 80.5 %, B6 93.3 %; spleen BALB/c 88.5 %, B6 50.8 %) and a higher percentage of lymphoid CD103(+) DCs in the spleen than females (BALB/c 41.5 %, B6 28.3 %). The percentage of NK cells was decreased in the spleen (BALB/c 12.5 %, B6 25.1 %) but increased in the PP (BALB/c 75.7 %, B6 78.6 %) of males as compared with females. Strain differences were also found in the PP; BALB/c mice had a higher percentage of T cells (males 58.1 %, females 75.5 %), a higher Th/Tc ratio (males 81.0 %, females 134.2 %), less FoxP3(+)CD25(-) T cells (males 14.6 %, females 30.0 %), more DCs (males 14.8 %, females 15.7 %) and macrophages (males 67.9 %, females 141.2 %), and more NK cells (males 160 %, females 164.3 %) than BALB/c mice. Conclusions: In this study, we show sex differences in intestinal and peripheral immune populations. These differences may underlie sex differences in intestinal disorders like IBD, and this information may be an important knowledge for the treatment of intestinal-related diseases

Loss of the Promyelocytic Leukemia Protein in Gastric Cancer: Implications for IP-10 Expression and Tumor-Infiltrating Lymphocytes

Gastric cancer is one of the most common causes of cancer-related mortality worldwide. Expression of the tumor suppressor, promyelocytic leukemia (PML) protein, is reduced or abolished in gastric carcinomas, in association with an increased level of lymphatic invasion, development of higher pTNM staging, and unfavorable prognosis. Herein, we investigated the relationship between the extent of tumor-infiltrating lymphocytes and the status of PML protein expression in advanced gastric carcinoma. We observed higher numbers of infiltrating T-cells in gastric carcinoma tissues in which PML expression was reduced or abolished, compared to tissues positive for PML. The extent of T-cell migration toward culture supernatants obtained from interferon-gamma (IFN-γ-stimulated gastric carcinoma cell lines was additionally affected by expression of PML in vitro. Interferon-gamma-inducible protein 10 (IP-10/CXCL10) expression was increased in gastric carcinoma tissues displaying reduced PML levels. Moreover, both Pml knockout and knockdown cells displayed enhanced IP-10 mRNA and protein expression in the presence of IFN-γ. PML knockdown increased IFN-γ-mediated Signal Transducer and Activator of Transcription-1 (STAT-1) binding to the IP-10 promoter, resulting in elevated transcription of the IP-10 gene. Conversely, PML IV protein expression suppressed IP-10 promoter activation. Based on these results, we propose that loss of PML protein expression in gastric cancer cells contributes to increased IP-10 transcription via enhancement of STAT-1 activity, which, in turn, promotes lymphocyte trafficking within tumor regions

Critical role of the disintegrin metalloprotease ADAM17 for intestinal inflammation and regeneration in mice

The protease a disintegrin and metalloprotease (ADAM) 17 cleaves tumor necrosis factor (TNF), L-selectin, and epidermal growth factor receptor (EGF-R) ligands from the plasma membrane. ADAM17 is expressed in most tissues and is up-regulated during inflammation and cancer. ADAM17-deficient mice are not viable. Conditional ADAM17 knockout models demonstrated proinflammatory activities of ADAM17 in septic shock via shedding of TNF. We used a novel gene targeting strategy to generate mice with dramatically reduced ADAM17 levels in all tissues. The resulting mice called ADAM17ex/ex were viable, showed compromised shedding of ADAM17 substrates from the cell surface, and developed eye, heart, and skin defects as a consequence of impaired EGF-R signaling caused by failure of shedding of EGF-R ligands. Unexpectedly, although the intestine of unchallenged homozygous ADAM17ex/ex mice was normal, ADAM17ex/ex mice showed substantially increased susceptibility to inflammation in dextran sulfate sodium colitis. This was a result of impaired shedding of EGF-R ligands resulting in failure to phosphorylate STAT3 via the EGF-R and, consequently, in defective regeneration of epithelial cells and breakdown of the intestinal barrier. Besides regulating the systemic availability of the proinflammatory cytokine TNF, our results demonstrate that ADAM17 is needed for vital regenerative activities during the immune response. Thus, our mouse model will help investigate ADAM17 as a potential drug target

VEGF receptor signaling links inflammation and tumorigenesis in colitis-associated cancer

Inflammation drives expression of VEGFR2, which is expressed on and drives growth of tumor cells in colitis-associated cancer

Androgen-Regulated Transcriptional Control of Sialyltransferases in Prostate Cancer Cells

The expression of gangliosides is often associated with cancer progression. Sialyltransferases have received much attention in terms of their relationship with cancer because they modulate the expression of gangliosides. We previously demonstrated that GD1a production was high in castration-resistant prostate cancer cell lines, PC3 and DU145, mainly due to their high expression of β-galactoside α2,3-sialyltransferase (ST3Gal) II (not ST3Gal I), and the expression of both ST3Gals was regulated by NF-κB, mainly by RelB. We herein demonstrate that GD1a was produced in abundance in cancerous tissue samples from human patients with hormone-sensitive prostate cancers as well as castration-resistant prostate cancers. The expression of ST3Gal II was constitutively activated in castration-resistant prostate cancer cell lines, PC3 and DU145, because of the hypomethylation of CpG island in its promoter. However, in androgen-depleted LNCap cells, a hormone-sensitive prostate cancer cell line, the expression of ST3Gal II was silenced because of the hypermethylation of the promoter region. The expression of ST3Gal II in LNCap cells increased with testosterone treatment because of the demethylation of the CpG sites. This testosterone-dependent ST3Gal II expression was suppressed by RelB siRNA, indicating that RelB activated ST3Gal II transcription in the testosterone-induced demethylated promoter. Therefore, in hormone-sensitive prostate cancers, the production of GD1a may be regulated by androgen. This is the first report indicating that the expression of a sialyltransferase is transcriptionally regulated by androgen-dependent demethylation of the CpG sites in its gene promoter