Regulatory network analysis of Paneth cell and goblet cell enriched gut organoids using transcriptomics approaches.

The epithelial lining of the small intestine consists of multiple cell types, including Paneth cells and goblet cells, that work in cohort to maintain gut health. 3D in vitro cultures of human primary epithelial cells, called organoids, have become a key model to study the functions of Paneth cells and goblet cells in normal and diseased conditions. Advances in these models include the ability to skew differentiation to particular lineages, providing a useful tool to study cell type specific function/dysfunction in the context of the epithelium. Here, we use comprehensive profiling of mRNA, microRNA and long non-coding RNA expression to confirm that Paneth cell and goblet cell enrichment of murine small intestinal organoids (enteroids) establishes a physiologically accurate model. We employ network analysis to infer the regulatory landscape altered by skewing differentiation, and using knowledge of cell type specific markers, we predict key regulators of cell type specific functions: Cebpa, Jun, Nr1d1 and Rxra specific to Paneth cells, Gfi1b and Myc specific for goblet cells and Ets1, Nr3c1 and Vdr shared between them. Links identified between these regulators and cellular phenotypes of inflammatory bowel disease (IBD) suggest that global regulatory rewiring during or after differentiation of Paneth cells and goblet cells could contribute to IBD aetiology. Future application of cell type enriched enteroids combined with the presented computational workflow can be used to disentangle multifactorial mechanisms of these cell types and propose regulators whose pharmacological targeting could be advantageous in treating IBD patients with Crohn's disease or ulcerative colitis.

Mucosal vaccines and technology.

There is an urgent and unmet need to develop effective vaccines to reduce the global burden of infectious disease in both animals and humans, and in particular for the majority of pathogens that infect via mucosal sites. Here we summarise the impediments to developing mucosal vaccines and review the new and emerging technologies aimed at overcoming the lack of effective vaccine delivery systems that is the major obstacle to developing new mucosal vaccines.

Use of bioengineered human commensal gut bacteria-derived microvesicles for mucosal plague vaccine delivery and immunization.

Plague caused by the Gram-negative bacterium, Yersinia pestis, is still endemic in parts of the world today. Protection against pneumonic plague is essential to prevent the development and spread of epidemics. Despite this, there are currently no licensed plague vaccines in the western world. Here we describe the means of delivering biologically active plague vaccine antigens directly to mucosal sites of plague infection using highly stable microvesicles (outer membrane vesicles; OMVs) that are naturally produced by the abundant and harmless human commensal gut bacterium Bacteroides thetaiotaomicron (Bt). Bt was engineered to express major plague protective antigens in its OMVs, specifically Fraction 1 (F1) in the outer membrane and LcrV (V antigen) in the lumen, for targeted delivery to the gastrointestinal (GI) and respiratory tracts in a non-human primate (NHP) host. Our key findings were that Bt OMVs stably expresses F1 and V plague antigens, particularly the V antigen, in the correct, immunogenic form. When delivered intranasally V-OMVs elicited substantive and specific immune and antibody responses, both in the serum [immunoglobulin (Ig)G] and in the upper and lower respiratory tract (IgA); this included the generation of serum antibodies able to kill plague bacteria. Our results also showed that Bt OMV-based vaccines had many desirable characteristics, including: biosafety and an absence of any adverse effects, pathology or gross alteration of resident microbial communities (microbiotas); high stability and thermo-tolerance; needle-free delivery; intrinsic adjuvanticity; the ability to stimulate both humoral and cell-mediated immune responses; and targeting of primary sites of plague infection.

A holistic approach to healthy ageing: how can people live longer, healthier lives?

BACKGROUND: Although lifespan is increasing, there is no evidence to suggest that older people are experiencing better health in their later years than previous generations. Nutrition, at all stages of life, plays an important role in determining health and wellbeing. METHODS: A roundtable meeting of UK experts on nutrition and ageing considered key aspects of the diet-ageing relationship and developed a consensus position on the main priorities for research and public health actions that are required to help people live healthier lives as they age. RESULTS: The group consensus highlighted the requirement for a life course approach, recognising the multifactorial nature of the impact of ageing. Environmental and lifestyle influences at any life stage are modified by genetic factors and early development. The response to the environment at each stage of life can determine the impact of lifestyle later on. There are no key factors that act in isolation to determine patterns of ageing and it is a combination of environmental and social factors that drives healthy or unhealthy ageing. Too little is known about how contemporary dietary patterns and sedentary lifestyles will impact upon healthy ageing in future generations and this is a priority for future research. CONCLUSIONS: There is good evidence to support change to lifestyle (i.e. diet, nutrition and physical) activity in relation to maintaining or improving body composition, cognitive health and emotional intelligence, immune function and vascular health. Lifestyle change at any stage of life may extend healthy lifespan, although the impact of early changes appears to be greatest.

Review article: the human intestinal virome in health and disease.

BACKGROUND: The human virome consists of animal-cell viruses causing transient infections, bacteriophage (phage) predators of bacteria and archaea, endogenous retroviruses and viruses causing persistent and latent infections. High-throughput, inexpensive, sensitive sequencing methods and metagenomics now make it possible to study the contribution dsDNA, ssDNA and RNA virus-like particles make to the human virome, and in particular the intestinal virome. AIM: To review and evaluate the pioneering studies that have attempted to characterise the human virome and generated an increased interest in understanding how the intestinal virome might contribute to maintaining health, and the pathogenesis of chronic diseases. METHODS: Relevant virome-related articles were selected for review following extensive language- and date-unrestricted, electronic searches of the literature. RESULTS: The human intestinal virome is personalised and stable, and dominated by phages. It develops soon after birth in parallel with prokaryotic communities of the microbiota, becoming established during the first few years of life. By infecting specific populations of bacteria, phages can alter microbiota structure by killing host cells or altering their phenotype, enabling phages to contribute to maintaining intestinal homeostasis or microbial imbalance (dysbiosis), and the development of chronic infectious and autoimmune diseases including HIV infection and Crohn's disease, respectively. CONCLUSIONS: Our understanding of the intestinal virome is fragmented and requires standardised methods for virus isolation and sequencing to provide a more complete picture of the virome, which is key to explaining the basis of virome-disease associations, and how enteric viruses can contribute to disease aetiologies and be rationalised as targets for interventions.

A role for the pattern recognition receptor Nod2 in promoting recruitment of CD103+ dendritic cells to the colon in response to Trichuris muris infection.

The ability of the colon to generate an immune response to pathogens, such as the model pathogen Trichuris muris, is a fundamental and critical defense mechanism. Resistance to T. muris infection is associated with the rapid recruitment of dendritic cells (DCs) to the colonic epithelium via epithelial chemokine production. However, the epithelial-pathogen interactions that drive chemokine production are not known. We addressed the role of the cytosolic pattern recognition receptor Nod2. In response to infection, there was a rapid influx of CD103(+)CD11c(+) DCs into the colonic epithelium in wild-type (WT) mice, whereas this was absent in Nod2(-/-) animals. In vitro chemotaxis assays and in vivo experiments using bone marrow chimeras of WT mice reconstituted with Nod2(-/-) bone marrow and infected with T. muris demonstrated that the migratory function of Nod2(-/-) DCs was normal. Investigation of colonic epithelial cell (CEC) innate responses revealed a significant reduction in epithelial production of the chemokines CCL2 and CCL5 but not CCL20 by Nod2-deficient CECs. Collectively, these data demonstrate the importance of Nod2 in CEC responses to infection and the requirement for functional Nod2 in initiating host epithelial chemokine-mediated responses and subsequent DC recruitment and T-cell responses following infection.

Acid-shock of Campylobacter jejuni induces flagellar gene expression and host cell invasion.

The bacterial pathogen Campylobacter jejuni is the leading cause of foodborne gastroenteritis in the developed world, with the organism being transmitted by ingestion of contaminated and undercooked poultry. Exposure to acid is an inevitable stressor for C. jejuni during gastric passage, yet the effect of low pH on C. jejuni virulence is still poorly understood. Here, we investigate the effect of acid-shock on C. jejuni viability, gene expression and host-cell invasion. C. jejuni strain NCTC 11168 survived acid exposure at pH 3.5 and above for up to 30 min without a drop in viability, and this exposure induced the expression of flagellar genes transcribed from σ(54)-dependent promoters. Furthermore, acid-shock resulted in increased C. jejuni invasion of m-ICcl2 mouse small intestine crypt cells grown on transwells, but not when the cells were grown on flat-bottomed wells. This suggests that C. jejuni might be invading intestinal epithelial cells at the basolateral side, possibly after paracellular passage. We hypothesize that acid-shock prior to intestinal entry may serve as a signal that primes C. jejuni to express its virulence gene repertoire including flagellar motility genes, but this requires further study in the context of an appropriate colonization or disease model.

gammadeltaT cell-mediated regulation of chemokine producing macrophages during Listeria monocytogenes infection-induced inflammation.

Infection of gammadeltaT cell-deficient (TcRdelta-/-) mice with the intracellular bacterium Listeria monocytogenes (Lm) results in an exacerbated inflammatory response characterized by the accumulation of activated macrophages and necrotic liver lesions. Here we investigated whether changes in chemokine production by Lm-elicited macrophages contribute to this abnormal inflammatory response. In response to Lm infection, activated macrophages accumulate in the primary sites of infection in TcRdelta-/- mice and express high amounts of mRNA encoding the chemokines CCL3 (MIP-1alpha), CCL4 (MIP-1beta), CXCL2 (MIP-2) and CXCL10 (IP-10). In the infected tissues of TcRdelta-/- the number of chemokine-synthesizing macrophages was higher than in wild-type (WT) mice, with the amount of MIP-1alpha and MIP-1beta secreted by individual macrophages in the spleen of TcRdelta-/- mice also being significantly higher than in WT mice. By contrast, protease activity and NO production in individual splenic macrophages of Lm-infected TcRdelta-/- and WT mice were comparable. Pathogen-elicited macrophages in TcRdelta-/- mice also expressed high levels of the CCL3 and CCL4 receptor, CCR5. In macrophage-gammadeltaT cell co-cultures, chemokine-producing macrophages were killed by cytotoxic Vgamma1+ T cells in a Fas-FasL-dependent manner consistent with the high levels of chemokine-producing macrophages seen in infected TcRdelta-/- mice being due to the absence of Vgamma1+ T cells. Together these findings highlight the importance of gammadeltaT cells in regulating macrophage anti-microbial responses.

Characterisation of fractalkine/CX3CL1 and fractalkine receptor (CX3CR1) expression in abdominal aortic aneurysm disease.

OBJECTIVES: Fractalkine (CX3CL1) promotes adhesion and extravasation of leucocytes through interactions with fractalkine receptor (CX3CR1) expressed on CD56+/CD16+ NK cells and CD8+ T cells. The current study aims to test the hypothesis the CX3CL1-CX3CR1 interaction contributes to the inflammatory infiltrate in AAA tissue. DESIGN AND METHODS: Immunohistochemistry (IHC) was used to define expression of CX3CR1 in AAA tissue. Multi-parametric flow cytometry (FC) was used to determine CX3CR1 expression on T-cells (CD3+) and NK cells (CD56+) from AAA tissue and peripheral blood of AAA patients and healthy controls. Regulation of CX3CL1 expression by vascular endothelial (vEC) and smooth muscle cells (vSMC) was examined in vitro using primary cell cultures. RESULTS: CX3CR1+ cells were detected in 19/28 AAA tissue samples and predominately localised in the adventitia. PBMCs from patients with AAA demonstrated higher percentages of CX3CR1+ NK cells (60.0-88.6%) and T cells (7.5-39.4%) compared with healthy controls. Furthermore, the frequency of CX3CR1+ NK cells (91%) and T cells (94%) in inflammatory AAA tissue were higher than in atherosclerotic AAA tissue. The pro-inflammatory cytokine TNFalpha increased expression of fractalkine by vSMC and vEC. CONCLUSION: CX3CL1+ and CX3CR1+ cells are present in AAA disease and their interaction may contribute to the recruitment of inflammatory cells seen in AAA tissue.

Pulmonary dendritic cells and alveolar macrophages are regulated by gammadelta T cells during the resolution of S. pneumoniae-induced inflammation.

gammadelta T cells commonly associate with mucosal and epithelial sites, fulfilling a variety of immunoregulatory functions. While lung gammadelta T cells have well-characterized pro-inflammatory activity, their potential role in the resolution of lung inflammation has yet to be explored in any detail. Indeed, given the importance of minimizing inflammation, the cellular mechanisms driving the resolution of lung inflammation are poorly understood. Using a murine model of acute Streptococcus pneumoniae-mediated lung inflammation, we now show that resolution of inflammation following bacterial clearance is associated with a > 30-fold increase in gammadelta T-cell number. Although inflammation eventually resolves in TCR delta(-/-) mice, elevated numbers of alveolar macrophages and pulmonary dendritic cells, and the appearance of well-formed granulomas in lungs of TCR delta(-/-) mice, together indicated a role for gammadelta T cells in regulating mononuclear phagocyte number. Ex vivo, both alveolar macrophages and pulmonary dendritic cells were susceptible to lung gammadelta T cell-mediated cytotoxicity, the first demonstration of such activity against a dendritic cell population. These findings support a model whereby expansion of gammadelta T cells helps restore mononuclear phagocyte numbers to homeostatic levels, protecting the lung from the consequences of inappropriate inflammation.

Increased natural killer cell activity in patients with an abdominal aortic aneurysm.

BACKGROUND: Natural killer (NK) cells have an emerging role in the development of chronic disease and in the direction and maintenance of inflammatory responses. Abdominal aortic aneurysms (AAA) is a chronic inflammatory disorder of unknown aetiology. The aim was to investigate whether NK cells showed altered function in patients with an AAA. METHODS: The presence, phenotype and function of peripheral blood and tissue NK cells from patients with an AAA, peripheral vascular disease (PVD) and healthy age-sex-matched controls were assessed before and after surgery. RESULTS: Patients with an AAA had significantly higher (P < 0.010) percentages of peripheral blood NK cells (mean (95 per cent c.i.) 23.8 (2.6) per cent) than patients with PVD (17.4 (2.9) per cent) and control subjects (16.2 (2.8) per cent). The NK cells from patients with an AAA had increased cytotoxicity on a per cell basis towards both an NK-sensitive target cell line and human aortic smooth muscle cells. Increased NK cell proportions (22.7 (3.5) per cent) and cytotoxic activity, together with higher C-reactive protein values, persisted after successful AAA repair. CONCLUSION: These data support the hypothesis that increased NK cytotoxicity could be a contributing factor in the generation or potentiation of inflammation in patients with an AAA.

Engineering of the gut commensal bacterium Bacteroides ovatus to produce and secrete biologically active murine interleukin-2 in response to xylan.

AIMS: The aim of this work was to engineer a gut commensal bacterium, Bacteroidesovatus, to produce and secrete a biologically active cytokine in a regulated manner as a basis for novel immunotherapies for chronic gut disorders. METHODS AND RESULTS: Bacteroides ovatus was engineered to produce murine interleukin-2 (MuIL2) intracellularly in response to xylan in culture media by inserting the MuIL2 gene into the xylanase operon of the organism. A second strain was engineered to secrete MuIL2 by adding Bacteroides fragilis enterotoxin secretion signal sequence to the protein. The recombinant strains produced MuIL2 only in the presence of xylan as determined by ELISA of cell lysates and culture supernatants. The IL2-dependent cell line CTLL-2 was used to demonstrate that MuIL2 produced by both B. ovatus strains was biologically active. This activity could be blocked by an anti-IL2 neutralizing antibody. The xylan-inducible nature of this system was demonstrated by RT-PCR. CONCLUSIONS: Bacteroides ovatus was successfully engineered to produce and secrete biologically active MuIL2 in a xylan-inducible manner. SIGNIFICANCE AND IMPACT OF THE STUDY: The production and secretion of a biologically active mammalian protein by a member of the gut microflora could lead to the development of new long-term immunotherapies for inflammatory gut diseases.

Colonic epithelial cell mediated suppression of CD4 T cell activation.

BACKGROUND AND AIMS: As the first point of contact with enteric antigens, intestinal epithelial cells (IEC) may be key in regulating mucosal immune responses. We determined therefore if murine colonic epithelial cells (CEC) have tolerogenic or activating effects on CD4 T cells. METHODS: Using a novel CEC, macrophages, and CD4 T cell coculture system, mitogen and antigen specific responses of naïve and antigen primed CD4 T cells were assessed. RESULTS: Although a proportion of CEC express the costimulatory molecules B7.1, B7.2, CD40, and CD54, they were unable to promote mitogen or antigen driven activation of CD4 T cells, even in the presence of exogenous costimulatory signals. CD4 T cells cocultured with CEC were CD25lo and CD45RBlo and remained in the G1 phase of the cell cycle. CEC were also able to prevent CD4 T cell activation by professional antigen presenting cells. CEC mediated suppression of T cell activation was cell contact dependent and transforming growth factor beta independent. CONCLUSIONS: These observations suggest that CEC contribute to the maintenance of T cell tolerance in the gut by preventing inappropriate activation of CD4 T cells.

Colonic dendritic cells, intestinal inflammation, and T cell-mediated bone destruction are modulated by recombinant osteoprotegerin.

Autoimmune associated bone disease and intestinal inflammation are closely linked with deregulation and hyperactivation of autoreactive CD4 T cells. How these T cells are activated and mediate disease is not clear. Here we show that in the Interleukin 2-deficient mouse model of autoimmunity spontaneous osteopenia and colitis are caused by increased production of the ligand for receptor activator of NFkappaB (RANKL). RANKL acting via its receptor, receptor activator of NFkappaB (RANK), increases bone turnover and promotes intestinal dendritic cell (DC) survival in vivo. Modulation of RANKL-RANK interactions with exogenous recombinant osteoprotegerin (Fc-OPG) reverses skeletal abnormalities and reduces colitis by decreasing colonic DC numbers. This study identifies a common causal link between bone disease and intestinal inflammation and establishes the importance of DC in mediating colonic inflammation in vivo.

A requirement for IL-2/IL-2 receptor signaling in intrathymic negative selection.

The nature of the signals that influence thymocyte selection and determine the fate of CD4(+)8(+) (double positive) thymocytes remains unclear. Cytokines produced locally in the thymus may modulate signals delivered by TCR-MHC/peptide interactions and thereby influence the fate of double-positive thymocytes. Because the IL-2/IL-2R signaling pathway has been implicated in thymocyte and peripheral T cell survival, we investigated the possibility that IL-2/IL-2R interactions contribute to the deletion of self-reactive, Ag-specific thymocytes. By using nontransgenic and transgenic IL-2-sufficient and -deficient animal model systems, we have shown that during TCR-mediated thymocyte apoptosis, IL-2 protein is expressed in situ in the thymus, and apoptotic thymocytes up-regulate expression of IL-2RS: IL-2R(+) double-positive and CD4 single-positive thymocytes undergoing activation-induced cell death bind and internalize IL-2. IL-2-deficient thymocytes are resistant to TCR/CD3-mediated apoptotic death, which is overcome by providing exogenous IL-2 to IL-2(-/-) mice. Furthermore, disruption or blockade of IL-2/IL-2R interactions in vivo during Ag-mediated selection rescues some MHC class II-restricted thymocytes from apoptosis. Collectively, these findings provide evidence for the direct involvement of the IL-2/IL-2R signaling pathway in the deletion of Ag-specific thymocyte populations and suggest that CD4 T cell hyperplasia and autoimmunity in IL-2(-/-) mice is a consequence of ineffective deletion of self-reactive T cells.

Uptake and presentation of antigen to T cells by primary colonic epithelial cells in normal and diseased states.

BACKGROUND & AIMS: The immunoregulatory properties of primary colonic epithelial cells (CECs) have not been defined. The ability of CECs from wild-type and interleukin 2-deficient (IL-2(-/-)) mice to take up a complex protein antigen and present peptides via MHC molecules to T cells was assessed and contrasted with that of primary small intestinal epithelial cells (SIECs). METHODS: Uptake of fluorescein isothiocyanate (FITC)-labeled ovalbumin (FITC-OVA) by CECs and SIECs from wild-type and IL-2(-/-) mice was measured by flow cytometry. The effect of disrupting cytoskeleton organization and metabolic activity of CEC on antigen uptake was assessed. An OVA/I-A(b)-specific CD4(+) T-cell line transfected with an NFAT-lacZ reporter gene construct was used to evaluate the ability of CECs and SIECs as well as CECs from healthy and colitic IL-2(-/-) mice to present antigen to T cells. RESULTS: Uptake of FITC-OVA by CECs is concentration dependent, is not saturated at physiologic concentrations, and requires metabolically active cells. CECs from IL-2(-/-) mice take up significantly more antigen than those from wild-type mice. CECs are more efficient APCs than SIECs, and antigen-pulsed CECs from IL-2(-/-) mice induce the highest levels of T-cell activation. CONCLUSIONS: Primary CECs are efficient APCs for CD4 MHC class II-restricted T cells. Antigen uptake and presentation is up-regulated in animals prone to develop intestinal inflammation.

Downmodulation of the inflammatory response to bacterial infection by gammadelta T cells cytotoxic for activated macrophages.

Although gammadelta T cells are involved in the regulation of inflammation after infection, their precise function is not known. Intraperitoneal infection of T cell receptor (TCR)-delta(-/-) mice with the intracellular bacterium Listeria monocytogenes resulted in the development of necrotic foci in the livers. In contrast, the peritoneal cavities of infected TCR-delta(-/-) mice contained an accumulation of low density activated macrophages and a reduced percentage of macrophages undergoing apoptosis. gammadelta T cell hybridomas derived from mice infected with Listeria were preferentially stimulated by low density macrophages from peritoneal exudates of infected mice. Furthermore, primary splenic gammadelta T cells isolated from Listeria-infected mice were cytotoxic for low density macrophages in vitro, and cytotoxicity was inhibited in the presence of antibodies to the gammadelta TCR. These results demonstrate a novel interaction between gammadelta T cells and activated macrophages in which gammadelta T cells are stimulated by terminally differentiated macrophages to acquire cytotoxic activity and which, in turn, induce macrophage cell death. This interaction suggests that gammadelta T cells regulate the inflammatory response to infection with intracellular pathogens by eliminating activated macrophages at the termination of the response.

Primary murine small intestinal epithelial cells, maintained in long-term culture, are susceptible to rotavirus infection.

We describe a method for long-term culture of primary small intestinal epithelial cells (IEC) from suckling mice. IEC were digested from intestinal fragments as small intact units of epithelium (organoids) by using collagenase and dispase. IEC proliferated from organoids on a basement-membrane-coated culture surface and remained viable for 3 weeks. Cultured IEC had the morphologic and functional characteristics of immature enterocytes, notably sustained expression of cytokeratin and alkaline phosphatase. Few mesenchymal cells were present in the IEC cultures. IEC were also cultured from adult BALB/c mice and expressed major histocompatibility complex (MHC) class II antigens for at least 48 h in vitro. Primary IEC supported the growth of rhesus rotavirus (RRV) to a greater extent than a murine small intestinal cell line, m-IC(cl2). Cell-culture-adapted murine rotavirus strain EDIM infected primary IEC and m-IC(cl2) cells to a lesser extent than RRV. Wild-type EDIM did not infect either cell type. Long-term culture of primary murine small intestinal epithelial cells provides a method to study (i) virus-cell interactions, (ii) the capacity of IEC to act as antigen-presenting cells using a wide variety of MHC haplotypes, and (iii) IEC biology.