Catherine Pythoud

Interleukin-17 Is a Critical Mediator of Vaccine-Induced Reduction of Helicobacter Infection in the Mouse Model

BACKGROUND & AIMS Despite the proven ability of immunization to reduce Helicobacter infection in mouse models, the precise mechanism of protection has remained elusive. This study explores the possibility that interleukin (IL)-17 plays a role in the reduction of Helicobacter infection following vaccination of wild-type animals or in spontaneous reduction of bacterial infection in IL-10-deficient mice. METHODS In mice, reducing Helicobacter infection, the levels and source of IL-17 were determined and the role of IL-17 in reduction of Helicobacter infection was probed by neutralizing antibodies. RESULTS Gastric IL-17 levels were strongly increased in mice mucosally immunized with urease plus cholera toxin and challenged with Helicobacter felis as compared with controls (654 +/- 455 and 34 +/- 84 relative units for IL-17 messenger RNA expression [P < .01] and 6.9 +/- 8.4 and 0.02 +/- 0.04 pg for IL-17 protein concentration [P < .01], respectively). Flow cytometry analysis showed that a peak of CD4(+)IL-17(+) T cells infiltrating the gastric mucosa occurred in immunized mice in contrast to control mice (4.7% +/- 0.3% and 1.4% +/- 0.3% [P < .01], respectively). Gastric mucosa-infiltrating CD4(+)IL-17(+) T cells were also observed in IL-10-deficient mice that spontaneously reduced H felis infection (4.3% +/- 2.3% and 2% +/- 0.6% [P < .01], for infected and noninfected IL-10-deficient mice, respectively). In wild-type immunized mice, intraperitoneal injection of anti-IL-17 antibodies significantly inhibited inflammation and the reduction of Helicobacter infection in comparison with control antibodies (1 of 12 mice vs 9 of 12 mice reduced Helicobacter infection [P < .01], respectively). CONCLUSIONS IL-17 plays a critical role in the immunization-induced reduction of Helicobacter infection from the gastric mucosa.

Granulocyte‐macrophage colony‐stimulating factor elicits bone marrow‐derived cells that promote efficient colonic mucosal healing

Background: Granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) therapy is effective in treating some Crohns disease (CD) patients and protects mice from colitis induced by dextran sulfate sodium (DSS) administration. However, its mechanisms of action remain elusive. We hypothesized that GM‐CSF affects intestinal mucosal repair. Methods: DSS colitic mice were treated with daily pegylated GM‐CSF or saline and clinical, histological, and inflammatory parameters were kinetically evaluated. Further, the role of bone marrow‐derived cells in the impact of GM‐CSF therapy on DSS colitis was addressed using cell transfers. Results: GM‐CSF therapy reduced clinical signs of colitis and the release of inflammatory mediators. GM‐CSF therapy improved mucosal repair, with faster ulcer reepithelialization, accelerated hyperproliferative response of epithelial cells in ulcer‐adjacent crypts, and lower colonoscopic ulceration scores in GM‐CSF‐administered mice relative to untreated mice. We observed that GM‐CSF‐induced promotion of mucosal repair is timely associated with a reduction in neutrophil numbers and increased accumulation of CD11b+ monocytic cells in colon tissues. Importantly, transfer of splenic GM‐CSF‐induced CD11b+ myeloid cells into DSS‐exposed mice improved colitis, and lethally irradiated GM‐CSF receptor‐deficient mice reconstituted with wildtype bone marrow cells were protected from DSS‐induced colitis upon GM‐CSF therapy. Lastly, GM‐CSF‐induced CD11b+ myeloid cells were shown to promote in vitro wound repair. Conclusions: Our study shows that GM‐CSF‐dependent stimulation of bone marrow‐derived cells during DSS‐induced colitis accelerates colonic tissue repair. These data provide a putative mechanism for the observed beneficial effects of GM‐CSF therapy in Crohns disease. (Inflamm Bowel Dis 2010;)

Targeting carbonic anhydrase IX improves the anti-cancer efficacy of mTOR inhibitors

The inhibition of the mechanistic target of rapamycin complex 1 (mTORC1) by chemical inhibitors, such as rapamycin, has demonstrated anti-cancer activity in preclinical and clinical trials. Their efficacy is, however, limited and tumors eventually relapse through resistance formation. In this study, using two different cancer mouse models, we identify tumor hypoxia as a novel mechanism of resistance of cancer cells against mTORC1 inhibitors. Indeed, we show that the activity of mTORC1 is mainly restricted to the non-hypoxic tumor compartment, as evidenced by a mutually exclusive staining pattern of the mTORC1 activity marker pS6 and the hypoxia marker pimonidazole. Consequently, whereas rapamycin reduces cancer cell proliferation in non-hypoxic regions, it has no effect in hypoxic areas, suggesting that cancer cells proliferate independently of mTORC1 under hypoxia. Targeting the hypoxic tumor compartment by knockdown of carbonic anhydrase IX (CAIX) using short hairpin RNA or by chemical inhibition of CAIX with acetazolamide potentiates the anti-cancer activity of rapamycin. Taken together, these data emphasize that hypoxia impairs the anti-cancer efficacy of rapalogs. Therapeutic strategies targeting the hypoxic tumor compartment, such as the inhibition of CAIX, potentiate the efficacy of rapamycin and warrant further clinical evaluation.

The Self-Inactivating KamiCas9 System for the Editing of CNS Disease Genes

Neurodegenerative disorders are a major public health problem because of the high frequency of these diseases. Genome editing with the CRISPR/Cas9 system is making it possible to modify the sequence of genes linked to these disorders. We designed the KamiCas9 self-inactivating editing system to achieve transient expression of the Cas9 protein and high editing efficiency. In the first application, the gene responsible for Huntingtons disease (HD) was targeted in adult mouse neuronal and glial cells. Mutant huntingtin (HTT) was efficiently inactivated in mouse models of HD, leading to an improvement in key markers of the disease. Sequencing of potential off-targets with the constitutive Cas9 system in differentiated human iPSC revealed a very low incidence with only one site above background level. This off-target frequency was significantly reduced with the KamiCas9 system. These results demonstrate the potential of the self-inactivating CRISPR/Cas9 editing for applications in the context of neurodegenerative diseases.

PAR2 Promotes Vaccine-Induced Protection Against Helicobacter Infection in Mice

BACKGROUND & AIMS Protective immunization limits Helicobacter infection of mice by undetermined mechanisms. Protease-activated receptor 2 (PAR2) signaling is believed to regulate immune and inflammatory responses. We investigated the role of PAR2 in vaccine-induced immunity against Helicobacter infection. METHODS Immune responses against Helicobacter infection were compared between vaccinated PAR2-/- and wild-type (WT) mice. Bacterial persistence, gastric pathology, and inflammatory and cellular responses were assessed using the rapid urease test (RUT), histologic analyses, quantitative polymerase chain reaction, and flow cytometry, respectively. RESULTS Following vaccination, PAR2-/- mice did not have reductions in Helicobacter felis infection (RUT values were 0.01±0.01 for WT mice and 0.11±0.13 for PAR2-/- mice; P<.05). The vaccinated PAR2-/- mice had reduced inflammation-induced stomach tissue damage (tissue damage scores were 8.83±1.47 for WT mice and 4.86±1.35 for PAR2-/- mice; P<.002) and reduced T-helper (Th)17 responses, based on reduced urease-induced interleukin (IL)-17 secretion by stomach mononuclear cells (5182 ± 1265 pg/mL for WT mice and 350±436 pg/mL for PAR2-/- mice; P<.03) and reduced recruitment of CD4+ IL-17+ T cells into the gastric mucosa of PAR2-/- mice following bacterial challenge (3.7%±1.5% for WT mice and 2.6%±1.1% for PAR2-/- mice; P<.05). In vitro, H felis-stimulated dendritic cells (DCs) from WT mice induced greater secretion of IL-17 by ovalbumin-stimulated OT-II transgenic CD4+ T cells compared with DCs from PAR2-/- mice (4298±347 and 3230±779; P<.04), indicating that PAR2-/- DCs are impaired in priming of Th17 cells. Adoptive transfer of PAR2+/+ DCs into vaccinated PAR2-/- mice increased vaccine-induced protection (RUT values were 0.11±0.10 and 0.26±0.15 for injected and noninjected mice, respectively; P<.03). CONCLUSIONS PAR2 activates DCs to mediate vaccine-induced protection against Helicobacter infection in mice.

Acidic tumor microenvironment abrogates the efficacy of mTORC1 inhibitors

BackgroundBlocking the mechanistic target of rapamycin complex-1 (mTORC1) with chemical inhibitors such as rapamycin has shown limited clinical efficacy in cancer. The tumor microenvironment is characterized by an acidic pH which interferes with cancer therapies. The consequences of acidity on the anti-cancer efficacy of mTORC1 inhibitors have not been characterized and are thus the focus of our study.MethodsCancer cell lines were treated with rapamycin in acidic or physiological conditions and cell proliferation was investigated. The effect of acidity on mTORC1 activity was determined by Western blot. The anticancer efficacy of rapamycin in combination with sodium bicarbonate to increase the intratumoral pH was tested in two different mouse models and compared to rapamycin treatment alone. Histological analysis was performed on tumor samples to evaluate proliferation, apoptosis and necrosis.ResultsExposing cancer cells to acidic pH in vitro significantly reduced the anti-proliferative effect of rapamycin. At the molecular level, acidity significantly decreased mTORC1 activity, suggesting that cancer cell proliferation is independent of mTORC1 in acidic conditions. In contrast, the activation of mitogen-activated protein kinase (MAPK) or AKT were not affected by acidity, and blocking MAPK or AKT with a chemical inhibitor maintained an anti-proliferative effect at low pH. In tumor mouse models, the use of sodium bicarbonate increased mTORC1 activity in cancer cells and potentiated the anti-cancer efficacy of rapamycin. Combining sodium bicarbonate with rapamycin resulted in increased tumor necrosis, increased cancer cell apoptosis and decreased cancer cell proliferation as compared to single treatment.ConclusionsTaken together, these results emphasize the inefficacy of mTORC1 inhibitors in acidic conditions. They further highlight the potential of combining sodium bicarbonate with mTORC1 inhibitors to improve their anti-tumoral efficacy.

Rapid transgene expression in multiple precursor cell types of adult rat subventricular zone mediated by adeno-associated type 1 vectors.

The adult rat brain subventricular zone (SVZ) contains proliferative precursors that migrate to the olfactory bulb (OB) and differentiate into mature neurons. Recruitment of precursors constitutes a potential avenue for brain repair. We have investigated the kinetics and cellular specificity of transgene expression mediated by AAV2/1 vectors (i.e., adeno-associated virus type 2 pseudotyped with AAV1 capsid) in the SVZ. Self-complementary (sc) and single-stranded (ss) AAV2/1 vectors mediated efficient GFP expression, respectively, at 17 and 24 hr postinjection. Transgene expression was efficient in all the rapidly proliferating cells types, that is, Mash1(+) precursors (30% of the GFP(+) cells), Dlx2(+) neuronal progenitors (55%), Olig2(+) oligodendrocyte progenitors (35%), and doublecortin-positive (Dcx(+)) migrating cells (40%), but not in the slowly proliferating glial fibrillary acidic protein-positive (GFAP(+)) neural stem cell pool (5%). Because cell cycle arrest by wild-type and recombinant AAV has been described in primary cultures, we examined SVZ proliferative activity after vector injection. Indeed, cell proliferation was reduced immediately after vector injection but was normal after 1 month. In contrast, migration and differentiation of GFP(+) precursors were unaltered. Indeed, the proportion of Dcx(+) cells was similar in the injected and contralateral hemispheres. Furthermore, 1 month after vector injection into the SVZ, GFP(+) cells, found, as expected, in the OB granular cell layer, were mature GABAergic neurons. In conclusion, the rapid and efficient transgene expression in SVZ neural precursors mediated by scAAV2/1 vectors underlines their potential usefulness for brain repair via recruitment of immature cells. The observed transient precursor proliferation inhibition, not affecting their migration and differentiation, will likely not compromise this strategy.

Acidic pH reduces VEGF-mediated endothelial cell responses by downregulation of VEGFR-2; relevance for anti-angiogenic therapies

Anti-angiogenic treatments targeting the vascular endothelial growth factor or its receptors have shown clinical benefits. However, impact on long-term survival remains limited. Solid tumors display an acidic microenvironment that profoundly influences their biology. Consequences of acidity on endothelial cells and anti-angiogenic therapies remain poorly characterized and hence are the focus of this study. We found that exposing endothelial cells to acidic extracellular pH resulted in reduced cell proliferation and migration. Also, whereas VEGF increased endothelial cell proliferation and survival at pH 7.4, it had no effect at pH 6.4. Furthermore, in acidic conditions, stimulation of endothelial cells with VEGF did not result in activation of downstream signaling pathways such as AKT. At a molecular level, acidity significantly decreased the expression of VEGFR-2 by endothelial cells. Consequently, anti-angiogenic therapies that target VEGFR-2 such as sunitinib and sorafenib failed to block endothelial cell proliferation in acidic conditions. In vivo, neutralizing tumor acidity with sodium bicarbonate increased the percentage of endothelial cells expressing VEGFR-2 in tumor xenografts. Furthermore, combining sodium bicarbonate with sunitinib provided stronger anti-cancer activity than either treatment alone. Histological analysis showed that sunitinib had a stronger anti-angiogenic effect when combined with sodium bicarbonate. Overall, our results show that endothelial cells prosper independently of VEGF in acidic conditions partly as a consequence of decreased VEGFR-2 expression. They further suggest that strategies aiming to raise intratumoral pH can improve the efficacy of anti-VEGF treatments.

Reactivation of AKT signaling following treatment of cancer cells with PI3K inhibitors attenuates their antitumor effects.

Targeting the phosphatidylinositol-3-kinase (PI3K) is a promising approach in cancer therapy. In particular, PI3K blockade leads to the inhibition of AKT, a major downstream effector responsible for the oncogenic activity of PI3K. However, we report here that small molecule inhibitors of PI3K only transiently block AKT signaling. Indeed, treatment of cancer cells with PI3K inhibitors results in a rapid inhibition of AKT phosphorylation and signaling which is followed by the reactivation of AKT signaling after 48 h as observed by Western blot. Reactivation of AKT signaling occurs despite effective inhibition of PI3K activity by PI3K inhibitors. In addition, wortmannin, a broad range PI3K inhibitor, did not block AKT reactivation suggesting that AKT signals independently of PI3K. In a therapeutical perspective, combining AKT and PI3K inhibitors exhibit stronger anti-proliferative and pro-apoptotic effects compared to AKT or PI3K inhibitors alone. Similarly, in a tumor xenograft mouse model, concomitant PI3K and AKT blockade results in stronger anti-cancer activity compared with either blockade alone. This study shows that PI3K inhibitors only transiently inhibit AKT which limits their antitumor activities. It also provides the proof of concept to combine PI3K inhibitors with AKT inhibitors in cancer therapy.

AAV5-miHTT gene therapy demonstrates suppression of mutant huntingtin aggregation and neuronal dysfunction in a rat model of Huntington’s disease

Huntington’s disease (HD) is a fatal progressive neurodegenerative disorder caused by a mutation in the huntingtin (HTT) gene. To date, there is no treatment to halt or reverse the course of HD. Lowering of either total or only the mutant HTT expression is expected to have therapeutic benefit. This can be achieved by engineered micro (mi)RNAs targeting HTT transcripts and delivered by an adeno-associated viral (AAV) vector. We have previously showed a miHTT construct to induce total HTT knock-down in Hu128/21 HD mice, while miSNP50T and miSNP67T constructs induced allele-selective HTT knock-down in vitro. In the current preclinical study, the mechanistic efficacy and gene specificity of these selected constructs delivered by an AAV serotype 5 (AAV5) vector was addressed using an acute HD rat model. Our data demonstrated suppression of mutant HTT messenger RNA, which almost completely prevented mutant HTT aggregate formation, and ultimately resulted in suppression of DARPP-32-associated neuronal dysfunction. The AAV5-miHTT construct was found to be the most efficient, although AAV5-miSNP50T demonstrated the anticipated mutant HTT allele selectivity and no passenger strand expression. Ultimately, AAV5-delivered-miRNA-mediated HTT lowering did not cause activation of microglia or astrocytes suggesting no immune response to the AAV5 vector or therapeutic precursor sequences. These preclinical results suggest that using gene therapy to knock-down HTT may provide important therapeutic benefit for HD patients and raised no safety concerns, which supports our ongoing efforts for the development of an RNA interference-based gene therapy product for HD.