Ludovic Huot

NOD2-mediated dysbiosis predisposes mice to transmissible colitis and colorectal cancer

Instability in the composition of gut bacterial communities (dysbiosis) has been linked to common human intestinal disorders, such as Crohns disease and colorectal cancer. Here, we show that dysbiosis caused by Nod2 deficiency gives rise to a reversible, communicable risk of colitis and colitis-associated carcinogenesis in mice. Loss of either Nod2 or RIP2 resulted in a proinflammatory microenvironment that enhanced epithelial dysplasia following chemically induced injury. The condition could be improved by treatment with antibiotics or an anti-interleukin-6 receptor-neutralizing antibody. Genotype-dependent disease risk was communicable via maternally transmitted microbiota in both Nod2-deficient and WT hosts. Furthermore, reciprocal microbiota transplantation reduced disease risk in Nod2-deficient mice and led to long-term changes in intestinal microbial communities. Conversely, disease risk was enhanced in WT hosts that were recolonized with dysbiotic fecal microbiota from Nod2-deficient mice. Thus, we demonstrated that licensing of dysbiotic microbiota is a critical component of disease risk. Our results demonstrate that NOD2 has an unexpected role in shaping a protective assembly of gut bacterial communities and suggest that manipulation of dysbiosis is a potential therapeutic approach in the treatment of human intestinal disorders.

Nod-like receptor pyrin domain-containing protein 6 (NLRP6) controls epithelial self-renewal and colorectal carcinogenesis upon injury

The colonic epithelium self-renews every 3 to 5 d, but our understanding of the underlying processes preserving wound healing from carcinogenesis remains incomplete. Here, we demonstrate that Nod-like receptor pyrin domain-containing protein 6 (NLRP6) suppresses inflammation and carcinogenesis by regulating tissue repair. NLRP6 was primarily produced by myofibroblasts within the stem-cell niche in the colon. Although NLRP6 expression was lowered in diseased colon, NLRP6-deficient mice were highly susceptible to experimental colitis. Upon injury, NLRP6 deficiency deregulated regeneration of the colonic mucosa and processes of epithelial proliferation and migration. Consistently, absence of NLRP6 accelerated colitis-associated tumor growth in mice. A gene-ontology analysis on a whole-genome expression profiling revealed a link between NLRP6 and self-renewal of the epithelium. Collectively, the integrity of the epithelial barrier is preserved by NLRP6 that may be manipulated to develop drugs capable of preventing adenoma formation in inflammatory bowel diseases.

Transcriptomic and genetic studies identify IL-33 as a candidate gene for Alzheimer's disease

The only recognized genetic determinant of the common forms of Alzheimers disease (AD) is the ɛ4 allele of the apolipoprotein E gene (APOE). To identify new candidate genes, we recently performed transcriptomic analysis of 2741 genes in chromosomal regions of interest using brain tissue of AD cases and controls. From 82 differentially expressed genes, 1156 polymorphisms were genotyped in two independent discovery subsamples (n=945). Seventeen genes exhibited at least one polymorphism associated with AD risk, and following correction for multiple testing, we retained the interleukin (IL)-33 gene. We first confirmed that the IL-33 expression was decreased in the brain of AD cases compared with that of controls. Further genetic analysis led us to select three polymorphisms within this gene, which we analyzed in three independent case–control studies. These polymorphisms and a resulting protective haplotype were systematically associated with AD risk in non-APOE ɛ4 carriers. Using a large prospective study, these associations were also detected when analyzing the prevalent and incident AD cases together or the incident AD cases alone. These polymorphisms were also associated with less cerebral amyloid angiopathy (CAA) in the brain of non-APOE ɛ4 AD cases. Immunohistochemistry experiments finally indicated that the IL-33 expression was consistently restricted to vascular capillaries in the brain. Moreover, IL-33 overexpression in cellular models led to a specific decrease in secretion of the Aβ40 peptides, the main CAA component. In conclusion, our data suggest that genetic variants in IL-33 gene may be associated with a decrease in AD risk potentially in modulating CAA formation.

Dynamic Compression of Chondrocyte-Agarose Constructs Reveals New Candidate Mechanosensitive Genes

Articular cartilage is physiologically exposed to repeated loads. The mechanical properties of cartilage are due to its extracellular matrix, and homeostasis is maintained by the sole cell type found in cartilage, the chondrocyte. Although mechanical forces clearly control the functions of articular chondrocytes, the biochemical pathways that mediate cellular responses to mechanical stress have not been fully characterised. The aim of our study was to examine early molecular events triggered by dynamic compression in chondrocytes. We used an experimental system consisting of primary mouse chondrocytes embedded within an agarose hydrogel; embedded cells were pre-cultured for one week and subjected to short-term compression experiments. Using Western blots, we demonstrated that chondrocytes maintain a differentiated phenotype in this model system and reproduce typical chondrocyte-cartilage matrix interactions. We investigated the impact of dynamic compression on the phosphorylation state of signalling molecules and genome-wide gene expression. After 15 min of dynamic compression, we observed transient activation of ERK1/2 and p38 (members of the mitogen-activated protein kinase (MAPK) pathways) and Smad2/3 (members of the canonical transforming growth factor (TGF)-β pathways). A microarray analysis performed on chondrocytes compressed for 30 min revealed that only 20 transcripts were modulated more than 2-fold. A less conservative list of 325 modulated genes included genes related to the MAPK and TGF-β pathways and/or known to be mechanosensitive in other biological contexts. Of these candidate mechanosensitive genes, 85% were down-regulated. Down-regulation may therefore represent a general control mechanism for a rapid response to dynamic compression. Furthermore, modulation of transcripts corresponding to different aspects of cellular physiology was observed, such as non-coding RNAs or primary cilium. This study provides new insight into how chondrocytes respond to mechanical forces.

The Toxoplasma nuclear factor TgAP2XI‐4 controls bradyzoite gene expression and cyst formation

Toxoplasma gondii undergoes many phenotypic changes during its life cycle. The recent identification of AP2 transcription factors in T. gondii has provided a platform for studying the mechanisms controlling gene expression. In the present study, we report that a recombinant protein encompassing the TgAP2XI‐4 AP2 domain was able to specifically bind to a DNA motif using gel retardation assays. TgAP2XI‐4 protein is localized in the parasite nucleus throughout the tachyzoite life cycle in vitro, with peak expression occurring after cytokinesis. We found that the TgAP2XI‐4 transcript level was higher in bradyzoite cysts isolated from brains of chronically infected mice than in the rapidly replicating tachyzoites. A knockout of the TgAP2XI‐4 gene in both T. gondii virulent type I and avirulent type II strains reveals its role in modulating expression and promoter activity of genes involved in stage conversion of the rapidly replicating tachyzoites to the dormant cyst forming bradyzoites. Furthermore, mice infected with the type II KO mutants show a drastically reduced brain cyst burden. Thus, our results validate TgAP2XI‐4 as a novel nuclear factor that regulates bradyzoite gene expression during parasite differentiation and cyst formation.

Transcriptomic analysis in the leech Theromyzon tessulatum: involvement of cystatin B in innate immunity.

At the present time, there is little information on mechanisms of innate immunity in invertebrate groups other than insects, especially annelids. In the present study, we have performed a transcriptomic study of the immune response in the leech Theromyzon tessulatum after bacterial challenge, by a combination of differential display RT (reverse transcriptase)-PCR and cDNA microarrays. The results show relevant modulations concerning several known and unknown genes. Indeed, threonine deaminase, malate dehydrogenase, cystatin B, polyadenylate-binding protein and alpha-tubulin-like genes are up-regulated after immunostimulation. We focused on cystatin B (stefin B), which is an inhibitor of cysteine proteinases involved in the vertebrate immune response. We have cloned the full-length cDNA and named the T. tessulatum gene as Tt-cysb. Main structural features of cystatins were identified in the derived amino acid sequence of Tt-cysb cDNA; namely, a glycine residue in the N-terminus and a consensus sequence of Gln-Xaa-Val-Xaa-Gly (QXVXG) corresponding to the catalytic site. Moreover, Tt-cysb is the first cystatin B gene characterized in invertebrates. We have determined by in situ hybridization and immunocytochemistry that Tt-cysb is only expressed in large coelomic cells. In addition, this analysis confirmed that Tt-cysb is up-regulated after bacterial challenge, and that increased expression occurs only in coelomic cells. These data demonstrate that the innate immune response in the leech involves a cysteine proteinase inhibitor that is not found in ecdysozoan models, such as Drosophila melanogaster or Caenorhabditis elegans, and so underlines the great need for information about innate immunity mechanisms in different invertebrate groups.

Reversion of antibiotic resistance in Mycobacterium tuberculosis by spiroisoxazoline SMARt-420

Countering TB prodrug resistance The arsenal of antibiotics for treating tuberculosis (TB) contains many prodrugs, such as ethionamide, which need activation by normal metabolism to release their toxic effects. Ethionamide is potentiated by small molecules. Blondiaux et al. screened for more potent analogs and identified a lead compound called SMARt-420. This small molecule inactivates a TetR-like repressor, EthR2, and boosts ethionamide activation. SMARt-420 successfully promoted clearance of multidrug-resistant strains of Mycobacterium tuberculosis from the lungs of mice. Science, this issue p. 1206 Resistance to an antituberculosis drug can be reversed by small molecules that activate a cryptic enzymatic pathway. Antibiotic resistance is one of the biggest threats to human health globally. Alarmingly, multidrug-resistant and extensively drug-resistant Mycobacterium tuberculosis have now spread worldwide. Some key antituberculosis antibiotics are prodrugs, for which resistance mechanisms are mainly driven by mutations in the bacterial enzymatic pathway required for their bioactivation. We have developed drug-like molecules that activate a cryptic alternative bioactivation pathway of ethionamide in M. tuberculosis, circumventing the classic activation pathway in which resistance mutations have now been observed. The first-of-its-kind molecule, named SMARt-420 (Small Molecule Aborting Resistance), not only fully reverses ethionamide-acquired resistance and clears ethionamide-resistant infection in mice, it also increases the basal sensitivity of bacteria to ethionamide.

Genetic stability of the live attenuated Bordetella pertussis vaccine candidate BPZE1

Despite the extensive use of efficacious pertussis vaccines, Bordetella pertussis infections are still among the main causes for childhood morbidity and mortality. Severe pertussis occurs mostly in very young children, often too young to be sufficiently protected by current vaccines, which require several administrations in regimens that vary between countries. Since natural infection with B. pertussis is able to induce protection, we have developed the live attenuated B. pertussis vaccine strain BPZE1 that protects mice upon a single intranasal administration. This strain was obtained by genetically inactivating pertussis toxin via two point mutations in the ptx gene, by deleting dnt encoding dermonecrotic toxin, and by replacing the B. pertussis ampG gene by Escherichia coli ampG, resulting in the removal of tracheal cytotoxin. Here, we assessed the genetic stability of BPZE1 after 20 and 27 weeks of continuous passaging in vitro and in vivo, respectively. BPZE1 was passaged 20 times in vitro and 9 times in vivo in Balb/C mice. After these passages, 8 hemolytic colonies were analyzed by PCR for the absence of dnt and B. pertussis ampG and the presence of E. coli ampG, by DNA sequencing for the presence of the two ptx point mutations and by DNA microarrays for the global genomic stability. In addition, the protective capacity of BPZE1 was evaluated after the passages. No genetic or protective difference was detected between the passaged bacteria and non-passaged BPZE1, indicating that stability of the vaccine strain is not a concern for BPZE1 to be considered as an attenuated live vaccine against whooping cough.

Evidence for induction of the ornithine transcarbamylase expression in Alzheimer's disease

To more rapidly identify candidate genes located within chromosomal regions of interest defined by genome scan studies in Alzheimers disease (AD), we have developed a customized microarray containing all the ORFs (n=2741) located within nine of these regions. Levels of gene expression were assessed in total RNA from brain tissue of 12 controls and 12 AD patients. Of all genes showing differential expression, we focused on the ornithine transcarbamylase (OTC) gene on Xp21.1., a key enzyme of the urea cycle which we found to be expressed in AD brains but not in controls, as confirmed by RT–PCR. We also detected mRNA expression of all the other urea cycle enzymes in AD brains. Immunochemistry experiments revealed that the OTC expression was strictly restricted to vascular endothelial cells in brain. Furthermore, OTC activity was 880% increased in the CSF of probable AD cases compared with controls. We analysed the association of the OTC −389 G/A and −241 A/G promoter polymorphisms with the risk of developing AD. We observed that rare haplotypes may be associated with the risk of AD through a possible modulation of the methylation of the OTC promoter. In conclusion, our results suggest the involvement of a new pathway in AD brains involving the urea cycle.

Toxoplasma transcription factor TgAP2XI-5 regulates the expression of genes involved in parasite virulence and host invasion

Background: Gene regulation in apicomplexan parasites is poorly understood. Results: The plant-like nuclear factor TgAP2XI-5 is targeted at gene promoters, including those required for parasite virulence. Conclusion: TgAP2XI-5 is a novel DNA sequence-specific transcription factor of T. gondii. Significance: Identifying master regulators of virulence gene expression is crucial for understanding of pathogenicity of this pathogen. Gene regulation in apicomplexan parasites, a phylum containing important protozoan parasites such as Plasmodium and Toxoplasma, is poorly understood. The life cycle of Toxoplasma gondii is complex, with multiple proliferation and differentiation steps, of which tachyzoite proliferation is the most relevant to pathogenesis in humans and animals. Tachyzoites express invasion and virulence factors that are crucial for their survival and manipulation of host cell functions. The expression of those factors is tightly controlled during the tachyzoite cell cycle to permit their correct packaging in newly formed apical secretory organelles named micronemes and rhoptries in the daughter cells. However, little is known about the factors that control the expression of genes encoding the virulence factors present in these parasite-specific secretory organelles. We report that the plant-like nuclear factor TgAP2XI-5 targets more than 300 gene promoters and actively controls the transcription of these genes. Most of these target genes, including those that are essential for parasite virulence, showed a peak of expression in the S and M phases of the cell cycle. Furthermore, we identified the cis-regulatory element recognized by TgAP2XI-5 and demonstrated its ability to actively drive gene transcription. Our results demonstrated that TgAP2XI-5 is a novel DNA sequence-specific transcription factor associated with promoter activation. TgAP2XI-5 may regulate gene transcription of crucial virulence factors in T. gondii.