Preconditioning with cell-free DNA prevents DSS-colitis by promoting cell protective autophagy.

Presence of cell-free DNA (cfDNA) in sera of patients with inflammatory bowel diseases (IBD) is a long-known fact. The biological effect of cfDNA administration on cellular autophagy within normal and inflammatory circumstances remains unclear. In this study, the effects of intravenous cfDNA pretreatment on autophagy response were studied in dextran sulfate sodium (DSS)-induced acute experimental colitis. Selected proinflammatory cytokine and autophagy-related gene and protein expressions were compared with clinical and histological activity parameters, and with transmission electron microscopic evaluations. A single intravenous dose of cfDNA pretreatment with cfDNA from colitis exhibited beneficial response concerning the clinical and histological severity of DSS-colitis as compared with effects of normal cfDNA. Pretreatment with colitis-derived cfDNA substantially altered the gene and protein expression of several autophagy and inflammatory cytokine genes in a clinically favorable manner. Autophagy in splenocytes is also altered after colitis-derived cfDNA pretreatment. During the process of acute colitis, the subsequent inflammatory environment presumably results in changes of cfDNA with the potential to facilitate cell protective autophagy. Understanding the molecular mechanisms behind the impact of colitis-associated autophagy, and elucidating alterations of the interaction between autophagy and innate immunity caused by nucleic acids may provide further insight into the etiology of IBD. By targeting or modifying cfDNA, novel anti-inflammatory therapies may be developed.

Modified Genomic Self-DNA Influences In Vitro Survival of HT29 Tumor Cells via TLR9- and Autophagy Signaling.

In relation of immunobiology, the consequence of the crosstalk between TLR9-signaling and autophagy is poorly documented in HT29 cancer cells. To assess the TLR9-mediated biologic effects of modified self-DNA sequences on cell kinetics and autophagy response HT29 cells were incubated separately with intact genomic (g), hypermethylated (m), fragmented (f), and hypermethylated/fragmented (m/f) self-DNAs. Cell viability, apoptosis, cell proliferation, colonosphere-formation were determined. Moreover, the relation of TLR9-signaling to autophagy response was assayed by real-time RT-PCR, immunocytochemistry and transmission electron microscopy (TEM). After incubation with g-, m-, and m/f-DNAs cell viability and proliferation decreased, while apoptosis increased. F-DNA treatment resulted in an increase of cell survival. Methylation of self-DNA resulted in decrease of TLR9 expression, while it did not influence the positive effect of DNA fragmentation on MyD88 and TRAF6 overexpression, and TNFα downregulation. Fragmentation of DNA abrogated the positive effect of methylation on IRAK2, NFκB and IL-8 mRNA upregulations. In case of the autophagy genes and proteins, g- and f-DNAs caused significant upregulation of Beclin1, Atg16L1, and LC3B. According to TEM analyses, autophagy was present in each group of tumor cells, but to a varying degree. Incubation with m-DNA suppressed tumor cell survival by inducing features of apoptotic cell death, and activated mitophagy. F-DNA treatment enhanced cell survival, and activated macroautophagy and lipophagy. Colonospheres were only present after m-DNA incubation. Our data provided evidence for a close existing interplay between TLR9-signaling and the autophagy response with remarkable influences on cell survival in HT29 cells subjected to modified self-DNA treatments.

Association of hepatocyte-derived growth factor receptor/caudal type homeobox 2 co-expression with mucosal regeneration in active ulcerative colitis.

AIM: To characterize the regeneration-associated stem cell-related phenotype of hepatocyte-derived growth factor receptor (HGFR)-expressing cells in active ulcerative colitis (UC). METHODS: On the whole 38 peripheral blood samples and 38 colonic biopsy samples from 18 patients with histologically proven active UC and 20 healthy control subjects were collected. After preparing tissue microarrays and blood smears HGFR, caudal type homeobox 2 (CDX2), prominin-1 (CD133) and Musashi-1 conventional and double fluorescent immunolabelings were performed. Immunostained samples were digitalized using high-resolution Mirax Desk instrument, and analyzed with the Mirax TMA Module software. For semiquantitative counting of immunopositive lamina propria (LP) cells 5 fields of view were counted at magnification × 200 in each sample core, then mean ± SD were determined. In case of peripheral blood smears, 30 fields of view with 100 µm diameter were evaluated in every sample and the number of immunopositive cells (mean ± SD) was determined. Using 337 nm UVA Laser MicroDissection system at least 5000 subepithelial cells from the lamina propria were collected. Gene expression analysis of HGFR, CDX2, CD133, leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5), Musashi-1 and cytokeratin 20 (CK20) were performed in both laser-microdisscted samples and blood samples by using real time reverse transcription polymerase chain reaction (RT-PCR). RESULTS: By performing conventional and double fluorescent immunolabelings confirmed by RT-PCR, higher number of HGFR (blood: 6.7 ± 1.22 vs 38.5 ± 3.18; LP: 2.25 ± 0.85 vs 9.22 ± 0.65; P < 0.05), CDX2 (blood: 0 vs 0.94 ± 0.64; LP: 0.75 ± 0.55 vs 2.11 ± 0.75; P < 0.05), CD133 (blood: 1.1 ± 0.72 vs 8.3 ± 1.08; LP: 11.1 ± 0.85 vs 26.28 ± 1.71; P < 0.05) and Musashi-1 (blood and LP: 0 vs scattered) positive cells were detected in blood and lamina propria of UC samples as compared to controls. HGFR/CDX2 (blood: 0 vs 1 ± 0.59; LP: 0.8 ± 0.69 vs 2.06 ± 0.72, P < 0.05) and Musashi-1/CDX2 (blood and LP: 0 vs scattered) co-expressions were found in blood and lamina propria of UC samples. HGFR/CD133 and CD133/CDX2 co-expressions appeared only in UC lamina propria samples. CDX2, Lgr5 and Musashi-1 expressions in UC blood samples were not accompanied by CK20 mRNA expression. CONCLUSION: In active UC, a portion of circulating HGFR-expressing cells are committed to the epithelial lineage, and may participate in mucosal regeneration by undergoing mesenchymal-to-epithelial transition.

Contribution of TLR signaling to the pathogenesis of colitis-associated cancer in inflammatory bowel disease.

In the intestine a balance between proinflammatory and repair signals of the immune system is essential for the maintenance of intestinal homeostasis. The innate immunity ensures a primary host response to microbial invasion, which induces an inflammatory process to localize the infection and prevent systemic dissemination of pathogens. The key elements of this process are the germline encoded pattern recognition receptors including Toll-like receptors (TLRs). If pathogens cannot be eliminated, they may elicit chronic inflammation, which may be partly mediated via TLRs. Additionally, chronic inflammation has long been suggested to trigger tissue tumorous transformation. Inflammation, the seventh hallmark of cancer, may affect all phases of tumor development, and evade the immune system. Inflammation acts as a cellular stressor and may trigger DNA damage or genetic instability. Furthermore, chronic inflammation can provoke genetic mutations and epigenetic mechanisms that promote malignant cell transformation. Colorectal cancers in inflammatory bowel disease patients are considered typical examples of inflammation-related cancers. Although data regarding the role of TLRs in the pathomechanism of cancer-associated colitis are rather conflicting, functionally these molecules can be classified as "largely antitumorigenic" and "largely pro-tumorigenic" with the caveat that the underlying signaling pathways are mainly context (i.e., organ-, tissue-, cell-) and ligand-dependent.

Preconditioning with intravenous colitic cell-free DNA prevents DSS-colitis by altering TLR9-associated gene expression profile.

BACKGROUND: Presence of cell-free-circulating DNA (fcDNA) sequences in sera of patients with inflammatory bowel diseases (IBD) is a well-established phenomenon. Potential roles of fcDNA in diagnosis, prognosis and therapy monitoring of chronic inflammatory colonic disorders have already been examined, albeit its actual biological function still remains unclear. AIMS AND METHODS: In the present experiment, we studied the immunobiological effects of isolated fcDNA of normal and inflammatory origin administered intravenously to mice prior to induction of dextran sulfate sodium (DSS)-colitis. In addition to evaluate the current disease and histological activity, changes of the gene expression profile in isolated lamina propria cells upon TLR9 ligation were assayed. RESULTS: A single intravenous dose of fcDNA pretreatment with colitic fcDNA exhibited beneficial response concerning the clinical and histological severity of DSS-colitis as compared to effects of normal fcDNA. Pretreatment with colitic fcDNA substantially altered the expression of several TLR9-related and inflammatory cytokine genes in a clinically favorable manner. CONCLUSIONS: During the process of acute colitis, the subsequent inflammatory environment presumably results in changes of fcDNA with the potential to facilitate the downregulation of inflammation and improvement of regeneration. Thus, preconditioning of mice with colitis-derived fcDNA via TLR9 signaling could exert a tissue-protective effect and influence beneficially the course of DSS-colitis. Elucidating mechanisms of immune response alterations by nucleic acids may provide further insight into the etiology of IBD and develop the basis of novel immunotherapies.

Interaction of autophagy and Toll-like receptors: a regulatory cross-talk--even in cancer cells?

Accumulating evidence indicates that the aberrantly altered process of autophagy is definitely involved in carcinogenesis. Nonetheless, Toll-like receptors (TLRs) sensing cell-derived pattern/danger-associated molecules also have the capacity to promote tumor development and immune escape. TLRs are usually expressed in immunocompetent cells, though several types of cancer cells have also been reported to display these innate immune receptors. On the other hand, however, both TLR- and autophagy-related signals may exert tumor suppressor mechanisms mainly in a cell-specific and context-dependent manner. The role of autophagy has been radically expanded, and now this machinery is considered as a fundamental eukaryotic cellular homeostatic process and integral component of the immune system influencing infection, inflammation and immunity. Recent studies have documented that TLRs and autophagy are interrelated in response to danger signals, furthermore there is a controling cross-talk among them to avoid deficient or excessive immunological effects. Although the potential interaction of autophagy and TLRs in cancer cells has not yet been clarified, it seems to be a critical aspect of cancer development and progression. Upon translation of basic knowledge into practice it is reasonable to speculate that modulation of the TLR-autophagy regulatory loop might be relevant for cancer treatment by providing further possible therapeutic targets.

Intravenous administration of a single-dose free-circulating DNA of colitic origin improves severe murine DSS-colitis.

In inflammatory bowel diseases the presence of free-circulating DNA (fcDNA) sequences in the sera is an established phenomenon, albeit its real biological function still remains unclear. In our study the immunobiologic effects of a single-dose, intravenously administered fcDNA of normal and colitic origin were assayed in DSS-colitic and control mice. In parallel with disease and histological activity evaluations changes of the TLR9 and inflammatory cytokine signaling gene expression profiles were assayed in isolated cells of the lamina propria. Intravenously administered colitis-derived fcDNA displayed a more prominent beneficial action regarding the clinical and histological severity of DSS-colitis than that of fcDNA of normal origin. Systemic administration of colitis-derived fcDNA significantly altered the expression of certain TLR9-related and proinflammatory cytokine genes in a clinically favorable manner. Presumably due to induction of severe colitis, the subsequent marked inflammatory environment may result changes in fcDNA with a potential to promote the downregulation of inflammation and improvement of tissue regeneration. Elucidating mechanisms of innate immune alterations by nucleic acids may provide further insight into the etiology of inflammatory bowel diseases, and develop the basis of novel nucleic acid-based immunotherapies.

Intratumoral functional heterogeneity and chemotherapy.

Intratumoral heterogeneity including genetic and nongenetic mechanisms refers to biological differences amongst malignant cells originated within the same tumor. Both, cell differentiation hierarchy and stochasticity in gene expression and signaling pathways may result in phenotypic differences of cancer cells. Since a tumor consists of cancer cell clones that display distinct behaviours, changes in clonal proliferative behavior may also contribute to the phenotypic variability of tumor cells. There is a need to reveal molecular actions driving chemotherapeutic resistance in colon cancer cells. In general, it is widely hypothesized that therapeutic resistance in colorectal cancer is a consequence of the preferential survival of cancer stem cells. However, recent data regarding colorectal cancer suggest that resistance to anticancer therapy and post-therapeutic tumor reappearence could be related to variations of clonal dynamics. Understanding the interaction of genetic and nongenetic determinants influencing the functional diversity and therapy response of tumors should be a future direction for cancer research.