The human gut microbiome as source of innovation for health: Which physiological and therapeutic outcomes could we expect?

From the moment of birth, each human being builds a microbe-host symbiosis which is key for the preservation of its health and well-being. This personal symbiotic coexistence is the result of progressive enrichments in microorganism diversity through external supplies. This diversity is nowadays massively overthrown by drastic changes related to clinical practice in birth management, environmental exposure, nutrition and healthcare behaviors. The last two generations have been the frame of massive modifications in life and food habits, with people being more and more sedentary, overfed and permeated with drugs and pollutants. We are now able to measure the impact of these changes on the gut microbiota diversity. Concomitantly, these modifications of lifestyle were associated with a dramatic increase in incidence of immune-mediated diseases including metabolic, allergic and inflammatory diseases and most likely neurodegenerative and psychiatric disorders. Microbiota is becoming a hot topic in the scientific community and in the mainstream media. The number of scientific publications increased by up to a factor three over the last five years, with gastrointestinal and metabolic diseases being the most productive areas. In the intellectual property landscape, the patent families on microbiota have more than doubled in the meantime. In parallel, funding either from National Institutes (e.g. from NIH which funds research mainly in the field of allergies, infections, cancer and cardiovascular diseases, from the White House which launched the national microbiome initiative) or by pharmaceutical companies follow the same trend, showing a boost and a strong support in the research field on microbiota. All major health players are investing in microbiome research as shown by the number of deals signed and by funding during 2015. The Giens round table addressed how the medicine of tomorrow, considering human beings as a human-microbe symbiotic supraorganism, could leverage microbiome knowledge and tools. The rationale for our working group has been structured around four domains of innovation that could derive from ongoing efforts in deciphering the interactions between human cells and intestinal microbiome as a central component of human health, namely: (1) development of stratification and monitoring tools; (2) identification of new target and drug discovery, as a part of our supra-genome; (4) exploitation of microbiota as a therapeutic target that can be modulated; (4) and finally as a source of live biotherapeutics and adjuvants. These four streams will exemplify how microbiota has changed the way we consider a wide range of chronic and incurable diseases and the consequences of long-lasting dysbiosis. In-depth microbiota analysis is opening one of the broadest fields of investigation for improving human and animal health and will be a source of major therapeutic innovations for tackling today's medical unmet needs. We thus propose a range of recommendations for basic researchers, care givers as well as for health authorities to gain reliability in microbiome analysis and accelerate discovery processes and their translation into applications for the benefits of the people. Finally, les Ateliers de Giens round table on microbiota benefited from the richness of the French ecosystem. France represents a center of excellence in the microbiota research field, with French institutions as Institut national de la recherche agronomique (INRA [Metagenopolis, Micalis]), Centre national de la recherché scientifique (CNRS), Unité de recherche sur les maladies infectieuses et tropicales émergentes (URMITE), Institut of Cardiometabolism and Nutrition (ICAN), Institut des maladies métaboliques et cardiovasculaires (I2MC), Institut national de la santé et de la recherche médicale (Inserm), Pasteur Institute and Gustave-Roussy being top-players for the number of publications.

Generation of a drug-inducible reporter system to study cell reprogramming in human cells.

BACKGROUND: Strategies on the basis of doxycycline-inducible lentiviruses in mouse cells allowed the examination of mechanisms governing somatic cell reprogramming. RESULTS: Using a doxycycline-inducible human reprogramming system, we identified unreported miRs enhancing reprogramming efficiency. CONCLUSION: We generated a drug-inducible human reprogramming reporter system as an invaluable tool for genetic or chemical screenings. SIGNIFICANCE: These cellular systems provide a tool to enable the advancement of reprogramming technologies in human cells. Reprogramming of somatic cells into induced pluripotent stem cells is achieved by the expression of defined transcription factors. In the last few years, reprogramming strategies on the basis of doxycycline-inducible lentiviruses in mouse cells became highly powerful for screening purposes when the expression of a GFP gene, driven by the reactivation of endogenous stem cell specific promoters, was used as a reprogramming reporter signal. However, similar reporter systems in human cells have not been generated. Here, we describe the derivation of drug-inducible human fibroblast-like cell lines that express different subsets of reprogramming factors containing a GFP gene under the expression of the endogenous OCT4 promoter. These cell lines can be used to screen functional substitutes for reprogramming factors or modifiers of reprogramming efficiency. As a proof of principle of this system, we performed a screening of a library of pluripotent-enriched microRNAs and identified hsa-miR-519a as a novel inducer of reprogramming efficiency.

Differential endothelial transcriptomics identifies semaphorin 3G as a vascular class 3 semaphorin.

OBJECTIVE: To characterize the role of a vascular-expressed class 3 semaphorin (semaphorin 3G [Sema3G]). METHODS AND RESULTS: Semaphorins have been identified as axon guidance molecules. Yet, they have more recently also been characterized as attractive and repulsive regulators of angiogenesis. Through a transcriptomic screen, we identified Sema3G as a molecule of angiogenic endothelial cells. Sema3G-deficient mice are viable and exhibit no overt vascular phenotype. Yet, LacZ expression in the Sema3G locus revealed intense arterial vascular staining in the angiogenic vasculature, starting at E9.5, which was detectable throughout adolescence and downregulated in adult vasculature. Sema3G is expressed as a full-length 100-kDa secreted molecule that is processed by furin proteases to yield 95- and a 65-kDa Sema domain-containing subunits. Full-length Sema3G binds to NP2, whereas processed Sema3G binds to NP1 and NP2. Expression profiling and cellular experiments identified autocrine effects of Sema3G on endothelial cells and paracrine effects on smooth muscle cells. CONCLUSIONS: Although the mouse knockout phenotype suggests compensatory mechanisms, the experiments identify Sema3G as a primarily endothelial cell-expressed class 3 semaphorin that controls endothelial and smooth muscle cell functions in autocrine and paracrine manners, respectively.

A cell-based drug delivery platform for treating central nervous system inflammation.

Mesenchymal stem cells (MSCs) are promising candidates for the development of cell-based drug delivery systems for autoimmune inflammatory diseases, such as multiple sclerosis (MS). Here, we investigated the effect of Ro-31-8425, an ATP-competitive kinase inhibitor, on the therapeutic properties of MSCs. Upon a simple pretreatment procedure, MSCs spontaneously took up and then gradually released significant amounts of Ro-31-8425. Ro-31-8425 (free or released by MSCs) suppressed the proliferation of CD4+ T cells in vitro following polyclonal and antigen-specific stimulation. Systemic administration of Ro-31-8425-loaded MSCs ameliorated the clinical course of experimental autoimmune encephalomyelitis (EAE), a murine model of MS, displaying a stronger suppressive effect on EAE than control MSCs or free Ro-31-8425. Ro-31-8425-MSC administration resulted in sustained levels of Ro-31-8425 in the serum of EAE mice, modulating immune cell trafficking and the autoimmune response during EAE. Collectively, these results identify MSC-based drug delivery as a potential therapeutic strategy for the treatment of autoimmune diseases. KEY MESSAGES: MSCs can spontaneously take up the ATP-competitive kinase inhibitor Ro-31-8425. Ro-31-8425-loaded MSCs gradually release Ro-31-8425 and exhibit sustained suppression of T cells. Ro-31-8425-loaded MSCs have more sustained serum levels of Ro-31-8425 than free Ro-31-8425. Ro-31-8425-loaded MSCs are more effective than MSCs and free Ro-31-8425 for EAE therapy.

A small-molecule screen for enhanced homing of systemically infused cells.

Poor homing of systemically infused cells to disease sites may limit the success of exogenous cell-based therapy. In this study, we screened 9,000 signal-transduction modulators to identify hits that increase mesenchymal stromal cell (MSC) surface expression of homing ligands that bind to intercellular adhesion molecule 1 (ICAM-1), such as CD11a. Pretreatment of MSCs with Ro-31-8425, an identified hit from this screen, increased MSC firm adhesion to an ICAM-1-coated substrate in vitro and enabled targeted delivery of systemically administered MSCs to inflamed sites in vivo in a CD11a- (and other ICAM-1-binding domains)-dependent manner. This resulted in a heightened anti-inflammatory response. This represents a new strategy for engineering cell homing to enhance therapeutic efficacy and validates CD11a and ICAM-1 as potential targets. Altogether, this multi-step screening process may significantly improve clinical outcomes of cell-based therapies.

Inhibition of the Ras-Net (Elk-3) pathway by a novel pyrazole that affects microtubules.

Net (Elk-3/SAP-2/Erp) is a transcription factor that is phosphorylated and activated by the Ras-extracellular signal-regulated kinase (Erk) signaling pathway and is involved in wound healing, angiogenesis, and tumor growth. In a cell-based screen for small molecule inhibitors of Ras activation of Net transcriptional activity, we identified a novel pyrazole, XRP44X. XRP44X inhibits fibroblast growth factor 2 (FGF-2)-induced Net phosphorylation by the Ras-Erk signaling upstream from Ras. It also binds to the colchicine-binding site of tubulin, depolymerizes microtubules, stimulates cell membrane blebbing, and affects the morphology of the actin skeleton. Interestingly, Combretastin-A4, which produces similar effects on the cytoskeleton, also inhibits FGF-2 Ras-Net signaling. This differs from other classes of agents that target microtubules, which have either little effect (vincristine) or no effect (docetaxel and nocodazole) on the Ras-Net pathway. XRP44X inhibits various cellular properties, including cell growth, cell cycle progression, and aortal sprouting, similar to other molecules that bind to the tubulin colchicine site. XRP44X has the potentially interesting property of connecting two important pathways involved in cell transformation and may thereby represent an interesting class of molecules that could be developed for cancer treatment.

The transcription factor Net regulates the angiogenic switch.

Angiogenesis is fundamental to physiological and pathological processes. Despite intensive efforts, little is known about the intracellular circuits that regulate angiogenesis. The transcription factor Net is activated by phosphorylation induced by Ras, an indirect regulator of angiogenesis. Net is expressed at sites of vasculogenesis and angiogenesis during early mouse development, suggesting that it could have a role in blood vessel formation. We show here that down-regulation of Net inhibits angiogenesis and vascular endothelial growth factor (VEGF) expression in vivo, ex vivo, and in vitro. Ras-activated phosphorylated Net (P-Net) stimulates the mouse VEGF promoter through the -80 to -53 region that principally binds Sp1. P-Net and VEGF are coexpressed in angiogenic processes in wild-type mouse tissues and in human tumors. We conclude that Net is a regulator of angiogenesis that can switch to an activator following induction by pro-angiogenic molecules.