Lynne Foster

IDO1 metabolites activate β-catenin signaling to promote cancer cell proliferation and colon tumorigenesis in mice.

BACKGROUND & AIMS Indoleamine 2,3 dioxygenase-1 (IDO1) catabolizes tryptophan along the kynurenine pathway. Although IDO1 is expressed in inflamed and neoplastic epithelial cells of the colon, its role in colon tumorigenesis is not well understood. We used genetic and pharmacologic approaches to manipulate IDO1 activity in mice with colitis-associated cancer and human colon cancer cell lines. METHODS C57Bl6 wild-type (control), IDO1-/-, Rag1-/-, and Rag1/IDO1 double-knockout mice were exposed to azoxymethane and dextran sodium sulfate to induce colitis and tumorigenesis. Colitis severity was assessed by measurements of disease activity, cytokine levels, and histologic analysis. In vitro experiments were conducted using HCT 116 and HT-29 human colon cancer cells. 1-methyl tryptophan and small interfering RNA were used to inhibit IDO1. Kynurenine pathway metabolites were used to simulate IDO1 activity. RESULTS C57Bl6 mice given pharmacologic inhibitors of IDO1 and IDO1-/- mice had lower tumor burdens and reduced proliferation in the neoplastic epithelium after administration of dextran sodium sulfate and azoxymethane than control mice. These reductions also were observed in Rag1/IDO1 double-knockout mice compared with Rag1-/- mice (which lack mature adaptive immunity). In human colon cancer cells, blockade of IDO1 activity reduced nuclear and activated β-catenin, transcription of its target genes (cyclin D1 and Axin2), and, ultimately, proliferation. Exogenous administration of IDO1 pathway metabolites kynurenine and quinolinic acid led to activation of β-catenin and proliferation of human colon cancer cells, and increased tumor growth in mice. CONCLUSIONS IDO1, which catabolizes tryptophan, promotes colitis-associated tumorigenesis in mice, independent of its ability to limit T-cell-mediated immune surveillance. The epithelial cell-autonomous survival advantage provided by IDO1 to colon epithelial cells indicate its potential as a therapeutic target.

The Bifunctional Entamoeba histolytica Alcohol Dehydrogenase 2 (EhADH2) Protein Is Necessary for Amebic Growth and Survival and Requires an Intact C-terminal Domain for Both Alcohol Dehydrogenase and Acetaldehyde Dehydrogenase Activity*

The intestinal protozoan pathogen Entamoeba histolytica lacks mitochondria and derives energy from the fermentation of glucose to ethanol with pyruvate, acetyl enzyme Co-A, and acetaldehyde as intermediates. A key enzyme in this pathway may be the 97-kDa bifunctional E. histolytica alcohol dehydrogenase 2 (EhADH2), which possesses both alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase activity (ALDH). EhADH2 appears to be a fusion protein, with separate N-terminal ALDH and C-terminal ADH domains. Here, we demonstrate that EhADH2 expression is required forE. histolytica growth and survival. We find that a mutant EhADH2 enzyme containing the C-terminal 453 amino acids of EhADH2 has ADH activity but lacks ALDH activity. However, a mutant consisting of the N-terminal half of EhADH2 possessed no ADH or ALDH activity. Alteration of a single histidine to arginine in the putative active site of the ADH domain eliminates both ADH and ALDH activity, and this mutant EhADH2 can serve as a dominant negative, eliminating both ADH and ALDH activity when co-expressed with wild-type EhADH2 inEscherichia coli. These data indicate that EhADH2 enzyme is required for E. histolytica growth and survival and that the C-terminal ADH domain of the enzyme functions as a separate entity. However, ALDH activity requires residues in both the N- and C-terminal halves of the molecule.

Antibodies to the serine rich Entamoeba histolytica protein (SREHP) prevent amoebic liver abscess in severe combined immunodeficient (SCID) mice

Amoebic liver abscess caused by Entamoeba histolytica is a major cause of morbidity and mortality worldwide. We used mice with severe combined immunodeficiency (SCID mice) to study the role of antibody in protection from amoebic liver abscess, and to identify protective antigens of E. histolytica. Antisera to recombinant versions of two major surface antigens of E. histolytica, the serine rich E. histolytica protein (SREHP) and the 170 kDa adhesin were used in this study. We found that 100% of SCID mice passively immunized with antiserum to the recombinant SREHP molecule were protected from developing amoebic liver abscess after intrahepatic challenge with virulent E. histolytica trophozoites. In contrast, preimmune serum, antiserum to a portion of the 170 kDa adhesin, and antiserum to the trpE fusion partner of SREHP did not protect SCID mice from amoebic liver abscess. Our study demonstrates that antibodies to a recombinant version of the amoebic SREHP molecule can protect against amoebic liver abscess, and suggest the recombinant SREHP molecule should be considered as a possible vaccine candidate to prevent amoebic liver abscess.

Hyaluronic acid is radioprotective in the intestine through a TLR4 and COX-2-mediated mechanism

The intestinal epithelium is sensitive to radiation injury. Damage to the intestinal epithelium is dose limiting in radiation therapy of abdominal cancers. There is a need for agents that can be given before radiation therapy to protect the intestinal epithelium. C57BL6 mice were subjected to 12 Gy of total body radiation. Some mice received intraperitoneal hyaluronic acid (HA) before radiation. Mice were killed 6 h after radiation to assess radiation-induced apoptosis in the intestine; other mice were killed at 84 h to assess crypt survival. Total body radiation (12 Gy) resulted in increased expression of HA synthases and HA in the intestine and increased plasma HA (5-fold). Intraperitoneal injection of HA (30 mg/kg) before radiation resulted in a 1.8-fold increase in intestinal crypt survival and a decrease in radiation-induced apoptosis. The radioprotective effects of HA were not seen in Toll-like receptor 4 (TLR4)- or cyclooxygenase-2 (COX-2)-deficient mice. Intraperitoneal injection of HA induced a 1.5-fold increase in intestinal COX-2 expression, a 1.5-fold increase in intestinal PGE₂, and the migration of COX-2-expressing mesenchymal stem cells from the lamina propria in the villi to the lamina propria near the crypt. We conclude that 1) radiation induces increased HA expression through inducing HA synthases, 2) intraperitoneal HA given before radiation reduces radiation-induced apoptosis and increases crypt survival, and 3) these radioprotective effects are mediated through TLR4, COX-2, and the migration of COX-2-expressing mesenchymal stem cells.

Immunogenicity of the recombinant serine rich Entamoeba histolytica protein (SREHP) amebiasis vaccine in the African Green Monkey

We report the first study in non-human primates of the safety and immunogenicity of a recombinant vaccine designed to prevent amebic liver abscess. In a pilot study, a recombinant vaccine containing the serine rich Entamoeba histolytica protein (SREHP) attached to a maltose binding protein (SREHP/MBP), which has been shown to be effective in preventing amebic liver abscess in rodent models of infection, was used to immunize two African Green Monkeys. Vaccination with SREHP/MBP resulted in no systemic side-effects. The monkeys receiving the SREHP/MBP protein developed antibodies that recognized the recombinant SREHP/MBP molecule, the native SREHP protein, and the surface of amebic trophozoites. Antiserum from SREHP/MBP-vaccinated monkeys could block the adhesion of E. histolytica trophozoites to mammalian cells, a feature that may correlate with vaccine efficacy. Attempts to produce amebic liver abscess in naive African Green Monkeys by direct hepatic inoculation with virulent E. histolytica trophozoites was not successful, suggesting this species is probably not suitable for vaccine efficacy studies.

Ibuprofen slows migration and inhibits bowel colonization by enteric nervous system precursors in zebrafish, chick and mouse.

Hirschsprung Disease (HSCR) is a potentially deadly birth defect characterized by the absence of the enteric nervous system (ENS) in distal bowel. Although HSCR has clear genetic causes, no HSCR-associated mutation is 100% penetrant, suggesting gene-gene and gene-environment interactions determine HSCR occurrence. To test the hypothesis that certain medicines might alter HSCR risk we treated zebrafish with medications commonly used during early human pregnancy and discovered that ibuprofen caused HSCR-like absence of enteric neurons in distal bowel. Using fetal CF-1 mouse gut slice cultures, we found that ibuprofen treated enteric neural crest-derived cells (ENCDC) had reduced migration, fewer lamellipodia and lower levels of active RAC1/CDC42. Additionally, inhibiting ROCK, a RHOA effector and known RAC1 antagonist, reversed ibuprofen effects on migrating mouse ENCDC in culture. Ibuprofen also inhibited colonization of Ret+/- mouse bowel by ENCDC in vivo and dramatically reduced bowel colonization by chick ENCDC in culture. Interestingly, ibuprofen did not affect ENCDC migration until after at least three hours of exposure. Furthermore, mice deficient in Ptgs1 (COX 1) and Ptgs2 (COX 2) had normal bowel colonization by ENCDC and normal ENCDC migration in vitro suggesting COX-independent effects. Consistent with selective and strain specific effects on ENCDC, ibuprofen did not affect migration of gut mesenchymal cells, NIH3T3, or WT C57BL/6 ENCDC, and did not affect dorsal root ganglion cell precursor migration in zebrafish. Thus, ibuprofen inhibits ENCDC migration in vitro and bowel colonization by ENCDC in vivo in zebrafish, mouse and chick, but there are cell type and strain specific responses. These data raise concern that ibuprofen may increase Hirschsprung disease risk in some genetically susceptible children.

CD44 and TLR4 mediate hyaluronic acid regulation of Lgr5+ stem cell proliferation, crypt fission and intestinal growth in postnatal and adult mice

Hyaluronic acid, a glycosaminoglycan in the extracellular matrix, binds to CD44 and Toll-like receptor 4 (TLR4). We previously addressed the role of hyaluronic acid in small intestinal and colonic growth in mice. We addressed the role of exogenous hyaluronic acid by giving hyaluronic acid intraperitoneally and the role of endogenous hyaluronic acid by giving PEP-1, a peptide that blocks hyaluronic acid binding to its receptors. Exogenous hyaluronic acid increased epithelial proliferation but had no effect on intestinal length. PEP-1 resulted in a shortened small intestine and colon and diminished epithelial proliferation. In the current study, we sought to determine whether the effects of hyaluronic acid on growth were mediated by signaling through CD44 or TLR4 by giving exogenous hyaluronic acid or PEP-1 twice a week from 3-8 wk of age to wild-type, CD44(-/-), and TLR4(-/-) mice. These studies demonstrated that signaling through both CD44 and TLR4 were important in mediating the effects of hyaluronic acid on growth in the small intestine and colon. Extending our studies to early postnatal life, we assessed the effects of exogenous hyaluronic acid and PEP-1 on Lgr5(+) stem cell proliferation and crypt fission. Administration of PEP-1 to Lgr5(+) reporter mice from postnatal day 7 to day 14 decreased Lgr5(+) cell proliferation and decreased crypt fission. These studies indicate that endogenous hyaluronic acid increases Lgr5(+) stem cell proliferation, crypt fission, and intestinal lengthening and that these effects are dependent on signaling through CD44 and TLR4.

Molecular analysis of carbohydrate antigen-induced monoclonal IgM anti-IgG antibodies (Rheumatoid factors)

Light and heavy chain variable regions of 11 monoclonal rheumatoid factors (MRF) produced after carbohydrate antigen immunization, and one MRF produced after protein immunization have been sequenced. Most carbohydrate antigen induced MRF utilized light chains that were homologous to light chains of MRF obtained from protein immune or LPS stimulated mice, and MRF derived from the autoimmune MRL/lpr mouse strain. VH gene usage was diverse for carbohydrate antigen induced MRF that bound all four isotypes of IgG, or that bound only the IgG3 isotype. In contrast VH gene use among our panel of MRF that bound the IgG1 isotype appeared restricted. Four of the five IgG1 binders used VH genes that were highly homologous to the VH nucleotide sequence of a gene encoding an NP binding monoclonal antibody. Our study confirms the use of a particular group of light chain genes among murine MRF, confirms that there is diversity in the heavy chain genes utilized among MRF, and suggests that a gene(s) homologous to the VH NP 23 J558 gene may be preferentially associated with murine MRF specificity for the IgG1 isotype.