Epithelial-specific A2B adenosine receptor signaling protects the colonic epithelial barrier during acute colitis.

Central to inflammatory bowel disease (IBD) pathogenesis is loss of mucosal barrier function. Emerging evidence implicates extracellular adenosine signaling in attenuating mucosal inflammation. We hypothesized that adenosine-mediated protection from intestinal barrier dysfunction involves tissue-specific signaling through the A2B adenosine receptor (Adora2b) at the intestinal mucosal surface. To address this hypothesis, we combined pharmacologic studies and studies in mice with global or tissue-specific deletion of the Adora2b receptor. Adora2b(-/-) mice experienced a significantly heightened severity of colitis, associated with a more acute onset of disease and loss of intestinal epithelial barrier function. Comparison of mice with Adora2b deletion on vascular endothelial cells (Adora2b(fl/fl)VeCadCre(+)) or intestinal epithelia (Adora2b(fl/fl)VillinCre(+)) revealed a selective role for epithelial Adora2b signaling in attenuating colonic inflammation. In vitro studies with Adora2b knockdown in intestinal epithelial cultures or pharmacologic studies highlighted Adora2b-driven phosphorylation of vasodilator-stimulated phosphoprotein (VASP) as a specific barrier repair response. Similarly, in vivo studies in genetic mouse models or treatment studies with an Adora2b agonist (BAY 60-6583) recapitulate these findings. Taken together, our results suggest that intestinal epithelial Adora2b signaling provides protection during intestinal inflammation via enhancing mucosal barrier responses.

Limited expression of APRIL and its receptors prior to intestinal IgA plasma cell development during human infancy.

The absence of immunoglobulin A (IgA) in the intestinal tract renders young infants highly susceptible to enteric infections. However, mediators of initial IgA induction in this population are undefined. We determined the temporal acquisition of plasma cells by isotype and expression of T cell-independent (TI) and -dependent (TD) IgA class switch factors in the human intestinal tract during early infancy. We found that IgA plasma cells were largely absent in the infant intestine until after 1 month of age, approaching adult densities later in infancy than both IgM and IgG. The restricted development of IgA plasma cells in the first month was accompanied by reduced expression of the TI factor a proliferation-inducing ligand (APRIL) and its receptors TACI (transmembrane activator and calcium-modulator and cyclophilin ligand interactor) and B cell maturation antigen (BCMA) within isolated lymphoid follicles (ILFs). Moreover, both APRIL and BCMA expression strongly correlated with increasing IgA plasma cell densities over time. Conversely, TD mediators (CD40 ligand (CD40L) and CD40) were expressed within ILFs before 1 month and were not associated with IgA plasma cell generation. In addition, preterm infants had lower densities of IgA plasma cells and reduced APRIL expression compared with full-term infants. Thus, blunted TI responses may contribute to the delayed induction of intestinal IgA during early human infancy.

Inhibition of N-terminal ATPase on HSP90 attenuates colitis through enhanced Treg function.

Inflammatory bowel disease (IBD) is a chronic inflammatory condition thought to reflect a failure of the enteral immune system to adequately regulate itself. Inflammatory stress drives upregulation of heat-shock proteins (HSPs), including the pro-inflammatory chaperone, HSP90. This protein sequesters the transcription factor, heat-shock factor 1 (HSF1) in the cytoplasm preventing transcription of a number of anti-inflammatory proteins. We hypothesized that inhibition of HSP90 would exert an anti-inflammatory effect and thereby attenuate intestinal inflammation in murine models of IBD. Inhibition of HSP90 with 17-allylaminogeldanamycin (17-AAG) reduced inflammation in acute dextran sodium sulfate and chronic CD45RB(High) colitis models coinciding with increased interleukin (IL)-10 production in the colon. Regulatory T cells (Tregs) from mice treated with 17-AAG demonstrated significantly greater suppressive capacity in vitro abolished in HSF1-/- or IL-10-/- cells. Finally, Tregs treated with 17-AAG exhibited increased nuclear localization of HSF1 with resultant upregulation of HSF1 response genes, including HSP70, HSP90 and IL-10.

An optimum anti-melanoma response in mice immunized with fibroblasts transfected with DNA from mouse melanoma cells requires the expression of both syngeneic and allogeneic MHC-determinants.

Most neoplasms do not induce antitumor immune responses that can control tumor growth. Tumor associated antigens (TAAs) are insufficiently immunogenic. A vaccine that augments the immunogenic properties of TAAs could be of importance in the treatment of cancer patients. In an animal model, we prepared a vaccine by transfection of highly antigenic allogeneic mouse fibroblasts (LM; H-2(k)) with DNA from B16 mouse melanoma cells. We then tested the transfected cells' immunogenic properties in C57BL/6 mice, syngeneic with the melanoma (H-2(b)). We hypothesized that the immunogenic properties of 'weak' TAAs formed by the neoplasm would be enhanced if they were expressed by highly antigenic cells. The results indicated that mice with melanoma treated by immunization with the DNA-transfected fibroblasts survived significantly longer than mice in various control groups. To investigate the contribution of MHC determinants expressed by the transfected cells to their immunogenic properties, we compared the antimelanoma responses in mice immunized with transfected cells that expressed allogeneic or syngeneic class I determinants. The results indicated that the immunogenic properties of the DNA-transfected cells were enhanced if the cells expressed allogeneic MHC determinants. The antimelanoma responses of greatest magnitude, however, mediated predominantly by CD8(+) T cells, were in mice immunized with transfected fibroblasts that expressed both syngeneic and allogeneic class I determinants.

Immunization with interleukin-2-secreting allogeneic cells transfected with DNA from mouse melanoma cells induces immune responses that prolong the lives of mice with melanoma.

Altered genes in tumor cells specify tumor-associated antigens. Because genetic instability is a characteristic of the malignant cell phenotype, a large number of different, altered genes may be present in a population of neoplastic cells, specifying an array of undefined tumor-associated determinants. We hypothesized that immunogenic cells transfected with DNA from malignant cells will include cells that specify tumor-associated antigens. To test this question, we deliberately mutagenized a population of B16 melanoma cells (H-2b) by ultraviolet-B irradiation. DNA from the surviving cells was used to transfect LM cells (H-2k), a mouse fibroblast cell line modified previously to secrete interleukin-2. The transfected allogeneic cells were then tested for their immunogenic properties in C57BL/6J mice (H-2b) syngeneic with the melanoma. Mice injected with a mixture of the mutagenized B16 cells and the transfected cells survived significantly longer than untreated mice injected with the mutagenized B16 cells alone. Mice injected with a mixture of mutagenized B16 cells and cells transfected with DNA from unirradiated B16 cells died in shorter intervals. Based on the results of cytotoxicity assays performed in vitro, the cellular immune responses of greatest magnitude were directed toward the type of cell from which the DNA was obtained.

Resistance to melanoma in mice immunized with semiallogeneic fibroblasts transfected with DNA from mouse melanoma cells.

Tumor-associated Ags (TAA) that characterize a population of malignant cells are recognized by CTLs in the context of determinants specified by the MHC class I locus. Nevertheless, most progressively growing neoplasms do not induce antitumor immune responses that can control tumor cell growth. The TAA may be insufficiently antigenic. We found previously that immunization of mice with a cellular immunogen prepared by transfecting tumor DNA into allogeneic mouse fibroblasts resulted in strong antitumor immune responses that were specific for the type of tumor from which the DNA was obtained. Since the fibroblasts differed at the MHC from the immunized mice, we postulated that the immunogenic properties of the allogeneic transfected cells might be enhanced if the cells were modified to express syngeneic class I determinants. In a mouse melanoma model system, the H-2Kb gene was introduced into LM mouse fibroblasts (H-2k). Afterward, the cells were transfected with DNA from B16 melanoma cells (H-2b). The transfected cells were tested for their immunotherapeutic properties in C57BL/6J mice (H-2b) with melanoma. Mice with melanoma treated solely by immunization with the semiallogeneic transfected cells developed strong, long-term resistance to the growth of the tumor. In some instances, the mice survived indefinitely. Intact rather than disrupted transfected cells were required to induce the antimelanoma response, consistent with direct presentation of TAA by the transfected cells. The augmented resistance to melanoma in mice treated with the semiallogeneic transfected cells points toward an analogous form of therapy for cancer patients.