Intestinal helminth infection drives carcinogenesis in colitis-associated colon cancer

Inflammatory bowel diseases (IBD) are chronic inflammatory disorders of the gastrointestinal tract, strongly associated with an increased risk of colorectal cancer development. Parasitic infections caused by helminths have been shown to modulate the host’s immune response by releasing immunomodulatory molecules and inducing regulatory T cells (Tregs). This immunosuppressive state provoked in the host has been considered as a novel and promising approach to treat IBD patients and alleviate acute intestinal inflammation. On the contrary, specific parasite infections are well known to be directly linked to carcinogenesis. Whether a helminth infection interferes with the development of colitis-associated colon cancer (CAC) is not yet known. In the present study, we demonstrate that the treatment of mice with the intestinal helminth Heligmosomoides polygyrus at the onset of tumor progression in a mouse model of CAC does not alter tumor growth and distribution. In contrast, H. polygyrus infection in the early inflammatory phase of CAC strengthens the inflammatory response and significantly boosts tumor development. Here, H. polygyrus infection was accompanied by long- lasting alterations in the colonic immune cell compartment, with reduced frequencies of colonic CD8+ effector T cells. Moreover, H. polygyrus infection in the course of dextran sulfate sodium (DSS) mediated colitis significantly exacerbates intestinal inflammation by amplifying the release of colonic IL-6 and CXCL1. Thus, our findings indicate that the therapeutic application of helminths during CAC might have tumor-promoting effects and therefore should be well-considered

Using a Specific RNA–Protein Interaction To Quench the Fluorescent RNA Spinach

RNAs are involved in interaction networks with other biomolecules and are crucial for proper cell function. Yet their biochemical analysis remains challenging. For Förster Resonance Energy Transfer (FRET), a common tool to study such interaction networks, two interacting molecules have to be fluorescently labeled. “Spinach” is a genetically encodable RNA aptamer that starts to fluoresce upon binding of an organic molecule. Therefore, it is a biological fluorophore tag for RNAs. However, spinach has never been used in a FRET assembly before. Here, we describe how spinach is quenched when close to acceptors. We used RNA–DNA hybridization to bring quenchers or red organic dyes in close proximity to spinach. Furthermore, we investigate RNA–protein interactions quantitatively on the example of <i>Pseudomonas aeruginosa</i> phage coat protein 7 (PP7) and its interacting pp7-RNA. We utilize spinach quenching as a fully genetically encodable system even under lysate conditions. Therefore, this work represents a direct method to analyze RNA–protein interactions by quenching the spinach aptamer

Covalent Attachment of Aggregation-Induced Emission Molecules to the Surface of Ultrasmall Gold Nanoparticles to Enhance Cell Penetration

Three different alkyne-terminated aggregation-induced emission molecules based on a para-substituted di-thioether were attached to the surface of ultrasmall gold nanoparticles (2 nm) by copper-catalyzed azide&ndash;alkyne cycloaddition (click chemistry). They showed a strong fluorescence and were well water-dispersible, in contrast to the dissolved AIE molecules. The AIE-loaded nanoparticles were not cytotoxic and easily penetrated the membrane of HeLa cells, paving the way for an intracellular application of AIE molecules, e.g., for imaging

Silencing of proinflammatory NF-κB and inhibition of herpes simplex virus (HSV) replication by ultrasmall gold nanoparticles (2 nm) conjugated with small-interfering RNA

Azide-terminated ultrasmall gold nanoparticles (2 nm gold core) were covalently functionalized with alkyne-terminated small-interfering siRNA duplexes by copper-catalyzed azide–alkyne cycloaddition (CuAAC; click chemistry). The nanoparticle core was visualized by transmission electron microscopy. The number of attached siRNA molecules per nanoparticle was determined by a combination of atomic absorption spectroscopy (AAS; for gold) and UV-Vis spectroscopy (for siRNA). Each nanoparticle carried between 6 and 10 siRNA duplex molecules which corresponds to a weight ratio of siRNA to gold of about 2.2 : 1. Different kinds of siRNA were conjugated to the nanoparticles, depending on the gene to be silenced. In general, the nanoparticles were readily taken up by cells and highly efficient in gene silencing, in contrast to free siRNA. This was demonstrated in HeLa-eGFP cells (silencing of eGFP) and in LPS-stimulated macrophages (silencing of NF-κB). Furthermore, we demonstrated that nanoparticles carrying antiviral siRNA potently inhibited the replication of Herpes simplex virus 2 (HSV-2) in vitro. This highlights the strong potential of siRNA-functionalized ultrasmall gold nanoparticles in a broad spectrum of applications, including gene silencing and treatment of viral infections, combined with a minimal dose of gold

Induction of Type I Interferons by Therapeutic Nanoparticle-Based Vaccination Is Indispensable to Reinforce Cytotoxic CD8+ T Cell Responses During Chronic Retroviral Infection

T cell dysfunction and immunosuppression are characteristic for chronic viral infections and contribute to viral persistence. Overcoming these burdens is the goal of new therapeutic strategies to cure chronic infectious diseases. We recently described that therapeutic vaccination of chronic retrovirus infected mice with a calcium phosphate (CaP) nanoparticle (NP)-based vaccine carrier, functionalized with CpG and viral peptides is able to efficiently reactivate the CD8+ T cell response and improve the eradication of virus infected cells. However, the mechanisms underlying this effect were largely unclear. While type I interferons (IFNs I) are considered to drive T cell exhaustion by persistent immune activation during chronic viral infection, we here describe an indispensable role of IFN I induced by therapeutic vaccination to efficiently reinforce cytotoxic CD8+ T cells (CTL) and improve control of chronic retroviral infection. The induction of IFN I is CpG dependent and leads to significant IFN signaling indicated by upregulation of IFN stimulated genes. By vaccinating chronically retrovirus-infected mice lacking the IFN I receptor (IFNAR−/−) or by blocking IFN I signaling in vivo during therapeutic vaccination, we demonstrate that IFN I signaling is necessary to drive full reactivation of CTLs. Surprisingly, we also identified an impaired suppressive capability of regulatory T cells in the presence of IFNα, which implicates an important role for vaccine-induced IFNα in the regulation of the T cell response during chronic retroviral infection. Our data suggest that inducing IFN I signaling in conjunction with the presentation of viral antigens can reactivate immune functions and reduce viral loads in chronic infections. Therefore, we propose CaP NPs as potential therapeutic tool to treat chronic infections

Colitis-associated colon cancer induction impacts the course of <i>H</i>. <i>polygyrus</i> infection.

<p>(A) Schematic time schedule of <i>H</i>. <i>polygyrus</i> (<i>H</i>.<i>poly</i>) infection and CAC induction in BALB/c mice. One day after i.p. injection of azoxymethane (AOM), mice were infected with 200 stage-three larvae (L3) <i>H</i>. <i>polygyrus</i>. After 6 days, dextran sulfate sodium (DSS) was given via the drinking water. DSS administration was repeated twice, and mice were analyzed at week 10 to 12. (B) Fecal egg counts were measured weekly during the course of the experiment until mice were sacrificed at week 10. Mice were graded as recovered when no eggs could be counted in their feces. The graph shows the percentage of recovered mice from 3 experiments with n = 10 <i>H</i>. <i>polygyrus</i> infected mice (open rectangles) and n = 19 CAC+<i>H</i>. <i>polygyrus</i> infected mice (grey rectangles) as survival curve. Statistical significance was calculated using log-rank test (*, p≤0.05). (C) At week 12, LPLs from the colons were isolated and stained for the expression of CD4 and intracellular IL-4. Frequencies of IL-4⁺ CD4⁺ T cells were determined by flow cytometry. (D) Colon biopsies were incubated <i>in vitro</i> for 6 hours in culture medium. Levels of IL-6 and CXCL1 in the supernatants were determined by Luminex and cytokines per milligram tissue were calculated. Bars represent the mean±SEM of data from 2–3 experiments (naïve, n = 4; naïve+<i>H</i>.<i>poly</i>, n = 6; CAC, n = 10–15; CAC+<i>H</i>.<i>poly</i>, n = 10–15). Statistical significance was calculated using one-way ANOVA followed by Dunn’s or Tukey's Multiple Comparison Test (*, p≤ 0.05; **, p≤ 0.01; ***, p≤ 0.001).</p

data_sheet_1_Induction of Type I Interferons by Therapeutic Nanoparticle-Based Vaccination Is Indispensable to Reinforce Cytotoxic CD8+ T Cell Responses During Chronic Retroviral Infection.PDF

<p>T cell dysfunction and immunosuppression are characteristic for chronic viral infections and contribute to viral persistence. Overcoming these burdens is the goal of new therapeutic strategies to cure chronic infectious diseases. We recently described that therapeutic vaccination of chronic retrovirus infected mice with a calcium phosphate (CaP) nanoparticle (NP)-based vaccine carrier, functionalized with CpG and viral peptides is able to efficiently reactivate the CD8⁺ T cell response and improve the eradication of virus infected cells. However, the mechanisms underlying this effect were largely unclear. While type I interferons (IFNs I) are considered to drive T cell exhaustion by persistent immune activation during chronic viral infection, we here describe an indispensable role of IFN I induced by therapeutic vaccination to efficiently reinforce cytotoxic CD8⁺ T cells (CTL) and improve control of chronic retroviral infection. The induction of IFN I is CpG dependent and leads to significant IFN signaling indicated by upregulation of IFN stimulated genes. By vaccinating chronically retrovirus-infected mice lacking the IFN I receptor (IFNAR^−/−) or by blocking IFN I signaling in vivo during therapeutic vaccination, we demonstrate that IFN I signaling is necessary to drive full reactivation of CTLs. Surprisingly, we also identified an impaired suppressive capability of regulatory T cells in the presence of IFNα, which implicates an important role for vaccine-induced IFNα in the regulation of the T cell response during chronic retroviral infection. Our data suggest that inducing IFN I signaling in conjunction with the presentation of viral antigens can reactivate immune functions and reduce viral loads in chronic infections. Therefore, we propose CaP NPs as potential therapeutic tool to treat chronic infections.</p

<i>H</i>. <i>polygyrus</i> infection in the late phase of colitis-associated colon cancer does not impact tumor growth.

<p>(A) Schematic time schedule of <i>H</i>. <i>polygyrus</i> (<i>H</i>.<i>poly</i>) infection and the induction of colitis-associated colon cancer (CAC) in BALB/c mice. After an intraperitoneal injection of the procarcinogen azoxymethane (AOM), 3 cycles of dextran sulfate sodium (DSS) were given via the drinking water. At week 8, mice were infected with 200 stage-three larvae (L3) <i>H</i>. <i>polygyrus</i> by oral gavage, and tumor development was analyzed at week 12. (B) Weight change of naïve mice (unfilled circles), AOM/DSS-treated mice (CAC, black circles) and AOM/DSS-treated <i>H</i>. <i>polygyrus</i> infected mice (CAC+<i>H</i>.<i>poly</i>, grey rectangles) relative to initial body weight during the course of the experiment. The graph shows data from 4 independent experiments (naïve, n = 12; CAC, n = 17; CAC+<i>H</i>.<i>poly</i>, n = 17). (C) Murine endoscopy was performed to obtain representative endoscopic images from the distal colon of CAC and CAC+<i>H</i>.<i>poly</i> mice and to determine tumor scores. Bars represent the mean±SEM of data from 4 experiments (CAC, n = 17; CAC+<i>H</i>.<i>poly</i>, n = 17). (D) Colon from naïve mice, <i>H</i>. <i>polygyrus</i> infected mice, CAC mice and CAC+<i>H</i>.<i>poly</i> mice were prepared to calculate colon weight to length ratios. Data from 4 independent experiments are shown (naïve, n = 12; naïve+<i>H</i>.<i>poly</i>, n = 16; CAC, n = 17; CAC+<i>H</i>.<i>poly</i>, n = 17). Statistical significance was calculated using one-way ANOVA followed by Tukey's Multiple Comparison Test (***, p≤ 0.001).</p

<i>H</i>. <i>polygyrus</i> infection leads to the expansion of regulatory T cells in the colon.

<p>BALB/c mice were infected with 200 stage-three larvae (L3) <i>H</i>. <i>polygyrus</i> by oral gavage, and at indicated time points post infection mice were sacrificed. (A) At day 14, representative tissue sections of the colon and the small intestine from naïve mice and <i>H</i>. <i>polygyrus</i> (<i>H</i>.<i>poly</i>) infected mice were fixed and stained with haematoxylin and eosin (H&E) or periodic acid Schiff (PAS) to show pathologic changes. Images show magnification at x200. (B) Goblet cells in PAS stained sections were counted and referred to villi length. Bars represent the mean±SEM of data from one experiment (naïve, n = 2; naïve+<i>H</i>.<i>poly</i>, n = 3). Statistical significance was calculated using unpaired t test (**, p≤ 0.01). (C) At indicated days post infection (dpi), colon from naïve mice and <i>H</i>. <i>polygyrus</i> infected mice were prepared and colon length was measured. Bars represent the mean±SEM of data from 2 independent experiments (naïve, n = 10; naïve+<i>H</i>.<i>poly</i>, n = 5). (D, E) At indicated dpi, mLNs and LPLs of naïve mice and <i>H</i>. <i>polygyrus</i> infected mice were isolated and stained for the expression of CD4, CD103 and intracellular Foxp3. Bars represent the mean±SEM of data from 3 independent experiments (naïve, n = 14; naïve+<i>H</i>.<i>poly</i>, n = 8). (F) To determine the suppressive capacity of Treg <i>in vitro</i>, Foxp3/eGFP mice were infected with 200 L3 <i>H</i>. <i>polygyrus</i>, and at day 10 post infection, CD4⁺eGFP⁺ (Foxp3⁺) T cells (Treg) from mLN of infected or naive mice were sorted. Tregs were co-cultured at a ratio of 1:1 with eFluor-labeled CD4⁺ responder T cells (Tresp) and with antigen-presenting cells in the presence of a-CD3. Proliferation of Tresp was measured by loss of eFluor dye. Bar diagram represents the proliferation as mean±SEM of 2 independent experiments (naïve, n = 7; <i>H</i>.<i>poly</i>, n = 7). Statistical significance was calculated using one-way ANOVA followed by Tukey's Multiple Comparison Test (*, p≤ 0.05; **, p≤ 0.01; ***, p≤ 0.001).</p

Gene silencing of IL-6 and CXCL1 ameliorates colonic inflammation in <i>H</i>. <i>polygyrus</i> infected DSS treated animals.

<p>(A) Schematic time schedule of <i>H</i>. <i>polygyrus</i> (<i>H</i>.<i>poly</i>) infection and siRNA treatment during the induction of dextran sulfate sodium colitis (DSS) in BALB/c mice. At day 1, mice were infected with 200 stage-three larvae (L3) <i>H</i>. <i>polygyrus</i> by oral gavage. Six days later, DSS was given via the drinking water for 7 days and mice were sacrificed on day 14. Eight μg siRNA-loaded CaP/PLGA nanoparticles directed against IL-6 and CXCL1 were applied intrarectally daily during DSS treatment until day 13. (B) Disease activity index (DAI) of DSS-treated mice (DSS, black circles), DSS-treated <i>H</i>. <i>polygyrus</i> infected mice (DSS+<i>H</i>.<i>poly</i>, grey rectangles) and siRNA+DSS-treated <i>H</i>. <i>polygyrus</i> infected mice (DSS+<i>H</i>.<i>poly</i>+siRNA, red rectangles) during the course of the experiment. The graph shows data from 1 experiment. Statistical significance was calculated using two-way ANOVA and Bonferroni posttests (**, p≤ 0.01). (C) Representative tissue sections of colon samples from DSS, DSS+<i>H</i>. <i>poly</i> and DSS+<i>H</i>.poly+siRNA mice were fixed and stained with haematoxylin and eosin to show pathologic changes. Images show magnification at x200. (D) At day 14 colons were prepared and biopsies from colon samples were cultured <i>in vitro</i> for 6 hours in culture medium. Levels of IL-6 and CXCL1 in the supernatants were determined by Luminex. Bars show the mean ± SEM of cytokines per milligram tissue from 1 experiment (DSS, n = 5; DSS+<i>H</i>.<i>poly</i>, n = 6; DSS+<i>H</i>.<i>poly</i>+siRNA, n = 5). Statistical significance was calculated using Mann Whitney test (*, p≤ 0.05).</p