Cell-Free Protein Synthesis by Diversifying Bacterial Transcription Machinery.

We have evaluated several approaches to increase protein synthesis in a cell-free coupled bacterial transcription and translation system. A strong pargC promoter, originally isolated from a moderate thermophilic bacterium Geobacillus stearothermophilus, was used to improve the performance of a cell-free system in extracts of Escherichia coli BL21 (DE3). A stimulating effect on protein synthesis was detected with extracts prepared from recombinant cells, in which the E. coli RNA polymerase subunits α, ß, ß' and ω are simultaneously coexpressed. Appending a 3' UTR genomic sequence and a T7 transcription terminator to the protein-coding region also improves the synthetic activity of some genes from linear DNA. The E. coli BL21 (DE3) rna::Tn10 mutant deficient in a periplasmic RNase I was constructed. The mutant cell-free extract increases by up to four-fold the expression of bacterial and human genes mediated from both bacterial pargC and phage pT7 promoters. By contrast, the RNase E deficiency does not affect the cell-free expression of the same genes. The regulatory proteins of the extremophilic bacterium Thermotoga, synthesized in a cell-free system, can provide the binding capacity to target DNA regions. The advantageous characteristics of cell-free systems described open attractive opportunities for high-throughput screening assays.

Intestinal epithelial Notch-1 protects from colorectal mucinous adenocarcinoma.

Increasing evidence links Notch-1 signaling with the maintenance of intestinal architecture and homeostasis. Dysfunction in the common Notch-1 pathway transcription factor recombinant binding protein suppressor of hairless (RBP-J) is associated with loss of epithelial barrier integrity and aberrant conversion of proliferative crypt cells into goblet cells. Furthermore, we have recently discovered that epithelial Notch-1 is indispensable in bridging innate and adaptive immunity in the gut and is required for supporting protective epithelial pro-inflammatory responses. Yet, the epithelial specific function of Notch-1 in intestinal tumorigenesis remains unknown. We generated Villin-Cre/Notch-1 fl/fl (VN -/- ) mice that are selectively deficient in Notch-1 in intestinal epithelial cells. Intestinal epithelial Notch-1 preserved barrier function and integrity, whereas lack of epithelial Notch-1 induced goblet cell hyperplasia, spontaneous serrated lesions, multifocal low- and high-grade dysplasia and colonic mucinous neoplasms in mice. Over time, VN -/- mice displayed high occurrence of colorectal mucinous adenocarcinomas, which correlated with increased levels of mitogenic, angiogenic and pro-tumorigenic gene expression. Finally, we found that the expression of Notch-1 is significantly reduced in human colorectal mucinous adenocarcinoma when compared to colorectal adenocarcinoma. Taken together, our findings reveal a novel and critical protective role for Notch-1 in controlling intestinal tumorigenesis.

CCDC88B is required for pathogenesis of inflammatory bowel disease.

Inflammatory bowel disease (IBD) involves interaction between host genetic factors and environmental triggers. CCDC88B maps within one IBD risk locus on human chromosome 11q13. Here we show that CCDC88B protein increases in the colon during intestinal injury, concomitant with an influx of CCDC88B+lymphoid and myeloid cells. Loss of Ccdc88b protects against DSS-induced colitis, with fewer pathological lesions and reduced intestinal inflammation in Ccdc88b-deficient mice. In a T cell transfer model of colitis, Ccdc88b mutant CD4+ T cells do not induce colitis in immunocompromised hosts. Expression of human CCDC88B RNA and protein is higher in IBD patient colons than in control colon tissue. In human CD14+ myeloid cells, CCDC88B is regulated by cis-acting variants. In a cohort of patients with Crohn's disease, CCDC88B expression correlates positively with disease risk. These findings suggest that CCDC88B has a critical function in colon inflammation and the pathogenesis of IBD.Hook-related protein family member CCDC88b is encoded by a locus that has been associated with inflammatory bowel disease. Here the authors show that Ccdc88b inactivation in T cells prevents colitis in a transfer model, and detect high colonic levels of CCDC88b in patients with Crohn disease or ulcerative colitis, identifying that expression correlates with disease risk.

STING Senses Microbial Viability to Orchestrate Stress-Mediated Autophagy of the Endoplasmic Reticulum.

Constitutive cell-autonomous immunity in metazoans predates interferon-inducible immunity and comprises primordial innate defense. Phagocytes mobilize interferon-inducible responses upon engagement of well-characterized signaling pathways by pathogen-associated molecular patterns (PAMPs). The signals controlling deployment of constitutive cell-autonomous responses during infection have remained elusive. Vita-PAMPs denote microbial viability, signaling the danger of cellular exploitation by intracellular pathogens. We show that cyclic-di-adenosine monophosphate in live Gram-positive bacteria is a vita-PAMP, engaging the innate sensor stimulator of interferon genes (STING) to mediate endoplasmic reticulum (ER) stress. Subsequent inactivation of the mechanistic target of rapamycin mobilizes autophagy, which sequesters stressed ER membranes, resolves ER stress, and curtails phagocyte death. This vita-PAMP-induced ER-phagy additionally orchestrates an interferon response by localizing ER-resident STING to autophagosomes. Our findings identify stress-mediated ER-phagy as a cell-autonomous response mobilized by STING-dependent sensing of a specific vita-PAMP and elucidate how innate receptors engage multilayered homeostatic mechanisms to promote immunity and survival after infection.

Epicutaneous Tolerance Induction to a Bystander Antigen Abrogates Colitis and Ileitis in Mice.

BACKGROUND: Although inflammatory bowel disease (IBD) is a failure in maintaining tolerance to the intestinal microbiota, few studies have investigated the use of immunologic tolerance as a treatment approach for IBD. We hypothesized that induction of immune tolerance at a distal site could suppress intestinal inflammation through a process of bystander regulation. METHODS: Epicutaneous tolerance was induced by topical application of ovalbumin (OVA) using a Viaskin patch for 48 hours. In some experiments, a single feed of ovalbumin was used to drive epicutaneous tolerance-induced regulatory T cells (Tregs) to the intestine. The mechanism of tolerance induction was tested using neutralizing antibodies against TGF-ß, IL-10, and Treg depletion using Foxp3-DTR mice. The capacity of skin-draining Tregs, or epicutaneous tolerance, to prevent or treat experimental IBD was tested using T-cell transfer colitis, dextran sodium sulfate (DSS) colitis, and ileitis in SAMP-YITFc mice. Weight loss, colonic inflammatory cytokines and histology were assessed. RESULTS: Epicutaneous exposure to ovalbumin induced systemic immune tolerance by a TGF-ß-dependent, but IL-10 and iFoxp3 Treg-independent mechanism. Skin draining Tregs suppressed the development of colitis. Epicutaneous tolerance to a model antigen prevented intestinal inflammation in the dextran sodium sulfate and SAMP-YITFc models and importantly could halt disease in mice already experiencing weight loss in the T-cell transfer model of colitis. This was accompanied by a significant accumulation of LAP and Foxp3 Tregs in the colon. CONCLUSIONS: This is the first demonstration that epicutaneous tolerance to a model antigen can lead to bystander suppression of inflammation and prevention of disease progression in preclinical models of IBD.

Dendritic cell-derived nitric oxide inhibits the differentiation of effector dendritic cells.

Dendritic cells (DCs) play a pivotal role in the development of effective immune defense while avoiding detrimental inflammation and autoimmunity by regulating the balance of adaptive immunity and immune tolerance. However, the mechanisms that govern the effector and regulatory functions of DCs are incompletely understood. Here, we show that DC-derived nitric oxide (NO) controls the balance of effector and regulatory DC differentiation. Mice deficient in the NO-producing enzyme inducible nitric oxide synthase (iNOS) harbored increased effector DCs that produced interleukin-12, tumor necrosis factor (TNF) and IL-6 but normal numbers of regulatory DCs that expressed IL-10 and programmed cell death-1 (PD-1). Furthermore, an iNOS-specific inhibitor selectively enhanced effector DC differentiation, mimicking the effect of iNOS deficiency in mice. Conversely, an NO donor significantly suppressed effector DC development. Furthermore, iNOS-/- DCs supported enhanced T cell activation and proliferation. Finally iNOS-/- mice infected with the enteric pathogen Citrobacter rodentium suffered more severe intestinal inflammation with concomitant expansion of effector DCs in colon and spleen. Collectively, our results demonstrate that DC-derived iNOS restrains effector DC development, and offer the basis of therapeutic targeting of iNOS in DCs to treat autoimmune and inflammatory diseases.

Critical function of the necroptosis adaptor RIPK3 in protecting from intestinal tumorigenesis.

Necroptosis is a programmed form of non-apoptotic cell death that requires the kinase activity of the receptor interacting protein kinase 3 (RIPK3). Although in vitro data suggests that cancer cells lacking expression of RIPK3 are invasive, the physiological role of RIPK3 in a disease-relevant setting remains unknown. Here we provide evidence that RIPK3 has a critical role in suppressing colorectal cancer (CRC). RIPK3-deficient mice were highly susceptible to colitis-associated CRC and exhibited greater production of pro-inflammatory mediators and tumor promoting factors. Tumorigenesis in RIPK3-deficiency resulted from uncontrolled activation of NF-κB, STAT3, AKT and Wnt-ß-catenin signaling pathways that enhanced the ability of intestinal epithelial cells (IECs) to aberrantly proliferate in the face of the sustained inflammatory microenvironment and promote CRC. We found that RIPK3 expression is reduced in tumors from patients with inflammatory bowel diseases, and further confirmed that expression of RIPK3 is downregulated in human CRC and correlated with cancer progression. Thus, our results reveal that the necroptosis adaptor RIPK3 has key anti-inflammatory and anti-tumoral functions in the intestine, and define RIPK3 as a novel colon tumor suppressor.

NOD-Like Receptors: Master Regulators of Inflammation and Cancer.

Cytosolic NOD-like receptors (NLRs) have been associated with human diseases including infections, cancer, and autoimmune and inflammatory disorders. These innate immune pattern recognition molecules are essential for controlling inflammatory mechanisms through induction of cytokines, chemokines, and anti-microbial genes. Upon activation, some NLRs form multi-protein complexes called inflammasomes, while others orchestrate caspase-independent nuclear factor kappa B (NF-κB) and mitogen activated protein kinase (MAPK) signaling. Moreover, NLRs and their downstream signaling components engage in an intricate crosstalk with cell death and autophagy pathways, both critical processes for cancer development. Recently, increasing evidence has extended the concept that chronic inflammation caused by abberant NLR signaling is a powerful driver of carcinogenesis, where it abets genetic mutations, tumor growth, and progression. In this review, we explore the rapidly expanding area of research regarding the expression and functions of NLRs in different types of cancers. Furthermore, we particularly focus on how maintaining tissue homeostasis and regulating tissue repair may provide a logical platform for understanding the liaisons between the NLR-driven inflammatory responses and cancer. Finally, we outline novel therapeutic approaches that target NLR signaling and speculate how these could be developed as potential pharmaceutical alternatives for cancer treatment.

NOD-like receptors in intestinal homeostasis and epithelial tissue repair.

The intestinal epithelium constitutes a dynamic physical barrier segregating the luminal content from the underlying mucosal tissue. Following injury, the epithelial integrity is restored by rapid migration of intestinal epithelial cells (IECs) across the denuded area in a process known as wound healing. Hence, through a sequence of events involving restitution, proliferation and differentiation of IECs the gap is resealed and homeostasis reestablished. Relapsing damage followed by healing of the inflamed mucosa is a hallmark of several intestinal disorders including inflammatory bowel diseases (IBD). While several regulatory peptides, growth factors and cytokines stimulate restitution of the epithelial layer after injury, recent evidence in the field underscores the contribution of innate immunity in controlling this process. In particular, nucleotide-binding and oligomerization domain-like receptors (NLRs) play critical roles in sensing the commensal microbiota, maintaining homeostasis, and regulating intestinal inflammation. Here, we review the process of intestinal epithelial tissue repair and we specifically focus on the impact of NLR-mediated signaling mechanisms involved in governing epithelial wound healing during disease.

Positional mapping and candidate gene analysis of the mouse Ccs3 locus that regulates differential susceptibility to carcinogen-induced colorectal cancer.

The Ccs3 locus on mouse chromosome 3 regulates differential susceptibility of A/J (A, susceptible) and C57BL/6J (B6, resistant) mouse strains to chemically-induced colorectal cancer (CRC). Here, we report the high-resolution positional mapping of the gene underlying the Ccs3 effect. Using phenotype/genotype correlation in a series of 33 AcB/BcA recombinant congenic mouse strains, as well as in groups of backcross populations bearing unique recombinant chromosomes for the interval, and in subcongenic strains, we have delineated the maximum size of the Ccs3 physical interval to a ∼2.15 Mb segment. This interval contains 12 annotated transcripts. Sequencing of positional candidates in A and B6 identified many either low-priority coding changes or non-protein coding variants. We found a unique copy number variant (CNV) in intron 15 of the Nfkb1 gene. The CNV consists of two copies of a 54 bp sequence immediately adjacent to the exon 15 splice site, while only one copy is found in CRC-susceptible A. The Nfkb1 protein (p105/p50) expression is much reduced in A tumors compared to normal A colonic epithelium as analyzed by immunohistochemistry. Studies in primary macrophages from A and B6 mice demonstrate a marked differential activation of the NfκB pathway by lipopolysaccharide (kinetics of stimulation and maximum levels of phosphorylated IκBα), with a more robust activation being associated with resistance to CRC. NfκB has been previously implicated in regulating homeostasis and inflammatory response in the intestinal mucosa. The interval contains another positional candidate Slc39a8 that is differentially expressed in A vs B6 colons, and that has recently been associated in CRC tumor aggressiveness in humans.

Intestinal antimicrobial peptides during homeostasis, infection, and disease.

Antimicrobial peptides (AMPs), including defensins and cathelicidins, constitute an arsenal of innate regulators of paramount importance in the gut. The intestinal epithelium is exposed to myriad of enteric pathogens and these endogenous peptides are essential to fend off microbes and protect against infections. It is becoming increasingly evident that AMPs shape the composition of the commensal microbiota and help maintain intestinal homeostasis. They contribute to innate immunity, hence playing important functions in health and disease. AMP expression is tightly controlled by the engagement of pattern recognition receptors (PRRs) and their impairment is linked to abnormal host responses to infection and inflammatory bowel diseases (IBD). In this review, we provide an overview of the mucosal immune barriers and the intricate crosstalk between the host and the microbiota during homeostasis. We focus on the AMPs and pay particular attention to how PRRs promote their secretion in the intestine. Furthermore, we discuss their production and main functions in three different scenarios, at steady state, throughout infection with enteric pathogens and IBD.

Effector functions of NLRs in the intestine: innate sensing, cell death, and disease.

Nucleotide-binding and oligomerization domain-like receptors (NLRs) are central regulators of pathogen recognition, the induction of innate immune effectors and inflammation with utmost importance in human diseases such as inflammatory bowel diseases. Most NLRs are key mediators of inflammasome complexes that activate caspase-1 and drive proteolytic processing of pro-inflammatory cytokines; however, a few tightly regulate inflammasome-independent activation of nuclear factor-κB and mitogen-activated protein kinase pathways. NLR signaling has evolved in intestinal epithelial cells to avoid overactive inflammatory responses toward the resident microbiota and to preserve epithelial barrier integrity and functions by maintaining homeostasis. In the present review, I examine new insights into the role of the NLRs in antimicrobial defenses. I pay particular attention to the emerging role of these receptors in engaging a complex cross talk between cell death and innate immunity pathways. Furthermore, I discuss the physiological functions of the NLRs in shaping the innate immune response within the intestine, maintaining homeostasis, inducing tissue repair following injury and promoting tumorigenesis.

Non-apoptotic role of BID in inflammation and innate immunity.

Innate immunity is a fundamental defence response that depends on evolutionarily conserved pattern recognition receptors for sensing infections or danger signals. Nucleotide-binding and oligomerization domain (NOD) proteins are cytosolic pattern-recognition receptors of paramount importance in the intestine, and their dysregulation is associated with inflammatory bowel disease. They sense peptidoglycans from commensal microorganisms and pathogens and coordinate signalling events that culminate in the induction of inflammation and anti-microbial responses. However, the signalling mechanisms involved in this process are not fully understood. Here, using genome-wide RNA interference, we identify candidate genes that modulate the NOD1 inflammatory response in intestinal epithelial cells. Our results reveal a significant crosstalk between innate immunity and apoptosis and identify BID, a BCL2 family protein, as a critical component of the inflammatory response. Colonocytes depleted of BID or macrophages from Bid(-/-) mice are markedly defective in cytokine production in response to NOD activation. Furthermore, Bid(-/-) mice are unresponsive to local or systemic exposure to NOD agonists or their protective effect in experimental colitis. Mechanistically, BID interacts with NOD1, NOD2 and the IκB kinase (IKK) complex, impacting NF-κB and extracellular signal-regulated kinase (ERK) signalling. Our results define a novel role of BID in inflammation and immunity independent of its apoptotic function, furthering the mounting evidence of evolutionary conservation between the mechanisms of apoptosis and immunity.

Caspase-12 dampens the immune response to malaria independently of the inflammasome by targeting NF-kappaB signaling.

Pathogen sensing by the inflammasome activates inflammatory caspases that mediate inflammation and cell death. Caspase-12 antagonizes the inflammasome and NF-κB and is associated with susceptibility to bacterial sepsis. A single-nucleotide polymorphism (T(125)C) in human Casp12 restricts its expression to Africa, Southeast Asia, and South America. Here, we investigated the role of caspase-12 in the control of parasite replication and pathogenesis in malaria and report that caspase-12 dampened parasite clearance in blood-stage malaria and modulated susceptibility to cerebral malaria. This response was independent of the caspase-1 inflammasome, as casp1(-/-) mice were indistinguishable from wild-type animals in response to malaria, but dependent on enhanced NF-κB activation. Mechanistically, caspase-12 competed with NEMO for association with IκB kinase-α/ß, effectively preventing the formation of the IκB kinase complex and inhibiting downstream transcriptional activation by NF-κB. Systemic inhibition of NF-κB or Ab neutralization of IFN-γ reversed the increased resistance of casp12(-/-) mice to blood-stage malaria infection.

Control of intestinal homeostasis, colitis, and colitis-associated colorectal cancer by the inflammatory caspases.

Inflammatory caspases are essential effectors of inflammation and cell death. Here, we investigated their roles in colitis and colorectal cancer and report a bimodal regulation of intestinal homeostasis, inflammation and tumorigenesis by caspases-1 and -12. Casp1(-/-) mice exhibited defects in mucosal tissue repair and succumbed rapidly after dextran sulfate sodium administration. This phenotype was rescued by administration of exogenous interleukin-18 and was partially reproduced in mice deficient in the inflammasome adaptor ASC. Casp12(-/-) mice, in which the inflammasome is derepressed, were resistant to acute colitis and showed signs of enhanced repair. Together with their increased inflammatory response, the enhanced repair response of Casp12(-/-) mice rendered them more susceptible to colorectal cancer induced by azoxymethane (AOM)+DSS. Taken together, our results indicate that the inflammatory caspases are critical in the induction of inflammation in the gut after injury, which is necessary for tissue repair and maintenance of immune tolerance.

Gender differences in expression of the human caspase-12 long variant determines susceptibility to Listeria monocytogenes infection.

Inflammatory caspases are important effectors of innate immunity. Caspase-12, of the inflammatory caspase subfamily, is expressed in all mammals tested to date, but has acquired deleterious mutation in humans. A single-nucleotide polymorphism introduces a premature stop codon in caspase-12 in the majority of the population. However, in 20% of African descendants, caspase-12 is expressed and sensitizes to infections and sepsis. Here, we examined the modalities by which human caspase-12 confers susceptibility to infection. We have generated a fully humanized mouse that expresses the human caspase-12 rare variant (Csp-12L) in a mouse casp-12(-/-) background. Characterization of the humanized mouse uncovered sex differences in Csp-12L expression and gender disparity in innate immunity to Listeria monocytogenes infection. The Csp-12L transgene completely reversed the knockout resistance-to-infection phenotype in casp-12(-/-) males. In contrast, it had a marginal effect on the response of female mice. We found that estrogen levels modulated the expression of caspase-12. Csp-12L was expressed in male mice but its expression was repressed in female mice. Administration of 17-beta-estradiol (E2) to humanized male mice had a direct suppressive effect on Csp-12L expression and conferred relative resistance to infection. Chromatin immunoprecipitation experiments revealed that caspase-12 is a direct transcriptional target of the estrogen receptor alpha (ERalpha) and mapped the estrogen response element (ERE) to intron 7 of the gene. We propose that estrogen-mediated inhibition of Csp-12L expression is a built-in mechanism that has evolved to protect females from infection.

Inflammasomes in infection and inflammation.

Two of the main challenges that multicellular organisms faced during evolution were to cope with invading microorganisms and eliminate and replace dying cells. Our innate immune system evolved to handle both tasks. Key aspects of innate immunity are the detection of invaders or tissue injury and the activation of inflammation that alarms the system through the action of cytokine and chemokine cascades. While inflammation is essential for host resistance to infections, it is detrimental when produced chronically or in excess and is linked to various diseases, most notably auto-immune diseases, auto-inflammatory disorders, cancer and septic shock. Essential regulators of inflammation are enzymes termed "the inflammatory caspases". They are activated by cellular sensors of danger signals, the inflammasomes, and subsequently convert pro-inflammatory cytokines into their mature active forms. In addition, they regulate non-conventional protein secretion of alarmins and cytokines, glycolysis and lipid biogenesis, and the execution of an inflammatory form of cell death termed "pyroptosis". By acting as key regulators of inflammation, energy metabolism and cell death, inflammatory caspases and inflammasomes exert profound influences on innate immunity and infectious and non-infectious inflammatory diseases.

Molecular regulation of inflammation and cell death.

Cell death and innate immunity are ancient evolutionary conserved processes that utilize a dazzling number of related molecular effectors and parallel signal transduction mechanisms. The investigation of the molecular mechanisms linking the sensing of a danger signal (pathogens or tissue damage) to the induction of an inflammatory response has witnessed a renaissance in the last few years. This was initiated by the identification of pattern recognition receptors (PRRs), including Toll-like receptors (TLRs) and more recently cytosolic Nod-like receptors (NLRs), that brought innate immunity to center stage and opened the field to the study of signal transduction pathways, adaptors and central effectors linked to PRRs. This led to the characterization of the inflammasome, a macromolecular complex, scaffolded by NLRs, that recruits and activates inflammatory caspases, which are essential effectors in inflammation and cell death responses. In this review, we describe the molecular pathways of cell death and innate immunity with a focus on recent advancements in both fields and an emphasis on the striking analogies between NLR innate immunity and mitochondrial apoptosis pathways.

Caspase-12 modulates NOD signaling and regulates antimicrobial peptide production and mucosal immunity.

Bacterial sensing by intracellular Nod proteins and other Nod-like receptors (NLRs) activates signaling pathways that mediate inflammation and pathogen clearance. Nod1 and Nod2 associate with the kinase Rip2 to stimulate NF-kappaB signaling. Other cytosolic NLRs assemble caspase-1-activating multiprotein complexes termed inflammasomes. Caspase-12 modulates the caspase-1 inflammasome, but unlike other NLRs, Nod1 and Nod2 have not been linked to caspases, and mechanisms regulating the Nod-Rip2 complex are less clear. We report that caspase-12 dampens mucosal immunity to bacterial infection independent of its effects on caspase-1. Caspase-12 deficiency enhances production of antimicrobial peptides, cytokines, and chemokines to entric pathogens, an effect dependent on bacterial type III secretion and the Nod pathway. Mechanistically, caspase-12 binds to Rip2, displacing Traf6 from the signaling complex, inhibiting its ubiquitin ligase activity, and blunting NF-kappaB activation. Nod activation and resulting antimicrobial peptide production constitute an early innate defense mechanism, and caspase-12 inhibits this mucosal antimicrobial response.