Aline Dupont

Dysbiotic gut microbiota causes transmissible Crohn's disease-like ileitis independent of failure in antimicrobial defence

Objectives Dysbiosis of the intestinal microbiota is associated with Crohns disease (CD). Functional evidence for a causal role of bacteria in the development of chronic small intestinal inflammation is lacking. Similar to human pathology, TNFdeltaARE mice develop a tumour necrosis factor (TNF)-driven CD-like transmural inflammation with predominant ileal involvement. Design Heterozygous TNFdeltaARE mice and wildtype (WT) littermates were housed under conventional (CONV), specific pathogen-free (SPF) and germ-free (GF) conditions. Microbial communities were analysed by high-throughput 16S ribosomal RNA gene sequencing. Metaproteomes were measured using LC-MS. Temporal and spatial resolution of disease development was followed after antibiotic treatment and transfer of microbial communities into GF mice. Granulocyte infiltration and Paneth cell function was assessed by immunofluorescence and gene expression analysis. Results GF-TNFdeltaARE mice were free of inflammation in the gut and antibiotic treatment of CONV-TNFdeltaARE mice attenuated ileitis but not colitis, demonstrating that disease severity and location are microbiota-dependent. SPF-TNFdeltaARE mice developed distinct ileitis-phenotypes associated with gradual loss of antimicrobial defence. 16S analysis and metaproteomics revealed specific compositional and functional alterations of bacterial communities in inflamed mice. Transplantation of disease-associated but not healthy microbiota transmitted CD-like ileitis to GF-TNFdeltaARE recipients and triggered loss of lysozyme and cryptdin-2 expression. Monoassociation of GF-TNFdeltaARE mice with the human CD-related Escherichia coli LF82 did not induce ileitis. Conclusions We provide clear experimental evidence for the causal role of gut bacterial dysbiosis in the development of chronic ileal inflammation with subsequent failure of Paneth cell function.

Properdin Plays a Protective Role in Polymicrobial Septic Peritonitis

Properdin is a positive regulator of complement activation so far known to be instrumental in the survival of infections with certain serotypes of Neisseria meningitidis. We have generated a fully backcrossed properdin-deficient mouse line by conventional gene-specific targeting. In vitro, properdin-deficient serum is impaired in alternative pathway-dependent generation of complement fragment C3b when activated by Escherichia coli DH5α. Properdin-deficient mice and wild-type littermates compare in their levels of C3 and IgM. In an in vivo model of polymicrobial septic peritonitis induced by sublethal cecal ligation and puncture, properdin-deficient mice appear immunocompromised, because they are significantly impaired in their survival compared with wild-type littermates. We further show that properdin localizes to mast cells and that properdin has the ability to directly associate with E. coli DH5α. We conclude that properdin plays a significant role in the outcome of polymicrobial sepsis.

MicroRNA-146a-mediated downregulation of IRAK1 protects mouse and human small intestine against ischemia/reperfusion injury.

Intestinal ischemia/reperfusion (I/R) injury causes inflammation and tissue damage and is associated with high morbidity and mortality. Uncontrolled activation of the innate immune system through toll‐like receptors (Tlr) plays a key role in I/R‐mediated tissue damage but the underlying mechanisms have not been fully resolved. Here, we identify post‐transcriptional upregulation of the essential Tlr signalling molecule interleukin 1 receptor‐associated kinase (Irak) 1 as the causative mechanism for post‐ischemic immune hyper‐responsiveness of intestinal epithelial cells. Increased Irak1 protein levels enhanced epithelial ligand responsiveness, chemokine secretion, apoptosis and mucosal barrier disruption in an experimental intestinal I/R model using wild‐type, Irak1−/− and Tlr4−/− mice and ischemic human intestinal tissue. Irak1 accumulation under hypoxic conditions was associated with reduced K48 ubiquitination and enhanced Senp1‐mediated deSUMOylation of Irak1. Importantly, administration of microRNA (miR)‐146a or induction of miR‐146a by the phytochemical diindolylmethane controlled Irak1 upregulation and prevented immune hyper‐responsiveness in mouse and human tissue. These findings indicate that Irak1 accumulation triggers I/R‐induced epithelial immune hyper‐responsiveness and suggest that the induction of miR‐146a offers a promising strategy to prevent I/R tissue injury.

Preclinical Investigations Reveal the Broad-Spectrum Neutralizing Activity of Peptide Pep19-2.5 on Bacterial Pathogenicity Factors

ABSTRACT Bacterial infections are known to cause severe health-threatening conditions, including sepsis. All attempts to get this disease under control failed in the past, and especially in times of increasing antibiotic resistance, this leads to one of the most urgent medical challenges of our times. We designed a peptide to bind with high affinity to endotoxins, one of the most potent pathogenicity factors involved in triggering sepsis. The peptide Pep19-2.5 reveals high endotoxin neutralization efficiency in vitro, and here, we demonstrate its antiseptic/anti-inflammatory effects in vivo in the mouse models of endotoxemia, bacteremia, and cecal ligation and puncture, as well as in an ex vivo model of human tissue. Furthermore, we show that Pep19-2.5 can bind and neutralize not only endotoxins but also other bacterial pathogenicity factors, such as those from the Gram-positive bacterium Staphylococcus aureus. This broad neutralization efficiency and the additive action of the peptide with common antibiotics makes it an exceptionally appropriate drug candidate against bacterial sepsis and also offers multiple other medication opportunities.

Age-dependent enterocyte invasion and microcolony formation by Salmonella.

The coordinated action of a variety of virulence factors allows Salmonella enterica to invade epithelial cells and penetrate the mucosal barrier. The influence of the age-dependent maturation of the mucosal barrier for microbial pathogenesis has not been investigated. Here, we analyzed Salmonella infection of neonate mice after oral administration. In contrast to the situation in adult animals, we observed spontaneous colonization, massive invasion of enteroabsorptive cells, intraepithelial proliferation and the formation of large intraepithelial microcolonies. Mucosal translocation was dependent on enterocyte invasion in neonates in the absence of microfold (M) cells. It further resulted in potent innate immune stimulation in the absence of pronounced neutrophil-dominated pathology. Our results identify factors of age-dependent host susceptibility and provide important insight in the early steps of Salmonella infection in vivo. We also present a new small animal model amenable to genetic manipulation of the host for the analysis of the Salmonella enterocyte interaction in vivo.

P. gingivalis in Periodontal Disease and Atherosclerosis – Scenes of Action for Antimicrobial Peptides and Complement

According to the NHS, it is estimated that over 50% of the adult population are, to some extent, affected by gum disease and approximately 15% of UK population have been diagnosed with severe periodontitis. Periodontitis, a chronic polymicrobial disease of the gums, causes inflammation in its milder form, whereas in its severe form affects the surrounding tissues and can result in tooth loss. During periodontitis, plaque accumulates and sits between the junctional epithelium and the tooth itself, resulting in inflammation and the formation of a periodontal pocket. An interface is formed directly between the subgingival bacteria and the junctional epithelial cells. Bacterial pathogens commonly associated with periodontal disease are, among others, Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola, together known as the “red complex.” This review will mostly concentrate on the role of P. gingivalis, a Gram-negative anaerobic bacterium and one of the major and most studied contributors of this disease. Because periodontal disease is associated with the development of atherosclerosis, it is important to understand the local immune response to P. gingivalis. Innate immune players, in particular, complement and antimicrobial peptides and their effects with regard to P. gingivalis during periodontitis and in the development of atherosclerosis will be presented.

Antimicrobial peptides and the enteric mucus layer act in concert to protect the intestinal mucosa

The intestinal mucosa squares the circle by allowing efficient nutrient absorption while generating a firm barrier toward the enteric microbiota, enteropathogenic microorganisms and high luminal concentrations of potent immunostimulatory molecules. The mucus layer together with local antimicrobial and anti-inflammatory peptides significantly contribute to this ability. Here we summarize the recent progress made to better understand the critical importance of this dynamic, complex and highly structured anti-inflammatory and antimicrobial barrier.

Bacterial cell wall compounds as Promising targets of antimicrobial agents I. Antimicrobial peptides and lipopolyamines

The first barrier that an antimicrobial agent must overcome when interacting with its target is the microbial cell wall. In the case of Gram-negative bacteria, additional to the cytoplasmic membrane and the peptidoglycan layer, an outer membrane (OM) is the outermost barrier. The OM has an asymmetric distribution of the lipids with phospholipids and lipopolysaccharide (LPS) located in the inner and outer leaflets, respectively. In contrast, Gram-positive bacteria lack OM and possess a much thicker peptidoglycan layer compared to their Gram-negative counterparts. An additional class of amphiphiles exists in Gram-positives, the lipoteichoic acids (LTA), which may represent important structural components. These long molecules cross-bridge the entire cell envelope with their lipid component inserting into the outer leaflet of the cytoplasmic membrane and the teichoic acid portion penetrating into the peptidoglycan layer. Furthermore, both classes of bacteria have other important amphiphiles, such as lipoproteins, whose importance has become evident only recently. It is not known yet whether any of these amphiphilic components are able to stimulate the immune system under physiological conditions as constituents of intact bacteria. However, all of them have a very high pro-inflammatory activity when released from the cell. Such a release may take place through the interaction with the immune system, or with antibiotics (particularly with those targeting cell wall components), or simply by the bacterial division. Therefore, a given antimicrobial agent must ideally have a double character, namely, it must overcome the bacterial cell wall barrier, without inducing the liberation of the pro-inflammatory amphiphiles. Here, new data are presented which describe the development and use of membrane-active antimicrobial agents, in particular antimicrobial peptides (AMPs) and lipopolyamines. In this way, essential progress was achieved, in particular with respect to the inhibition of deleterious consequences of bacterial infections such as severe sepsis and septic shock.

Intestinal mucus affinity and biological activity of an orally administered antibacterial and anti-inflammatory peptide

Objective Antimicrobial peptides (AMP) provide protection from infection by pathogenic microorganisms and restrict bacterial growth at epithelial surfaces to maintain mucosal homeostasis. In addition, they exert a significant anti-inflammatory activity. Here we analysed the anatomical distribution and biological activity of an orally administered AMP in the context of bacterial infection and host–microbial homeostasis. Design The anatomical distribution as well as antibacterial and anti-inflammatory activity of the endogenous AMP cryptdin 2 and the synthetic peptide Pep19-2.5 at the enteric mucosal surface were analysed by immunostaining, functional viability and stimulation assays, an oral Salmonella enterica subsp. enterica sv. Typhimurium (S. Typhimurium) model and comparative microbiota analysis. Results Endogenous cryptdin 2 was found attached to bacteria of the enteric microbiota within the intestinal mucus layer. Similarly, the synthetic peptide Pep19-2.5 attached rapidly to bacterial cells, exhibited a marked affinity for the intestinal mucus layer in vivo, altered the structural organisation of endotoxin in a mucus matrix and demonstrated potent anti-inflammatory and antibacterial activity. Oral Pep19-2.5 administration induced significant changes in the composition of the enteric microbiota as determined by high-throughput 16S rDNA sequencing. This may have contributed to the only transient improvement of the clinical symptoms after oral infection with S. Typhimurium. Conclusions Our findings demonstrate the anti-inflammatory activity and mucus affinity of the synthetic AMP Pep19-2.5 and characterise the influence on microbiota composition and enteropathogen infection after oral administration.

Bacterial Cell Wall Compounds as Promising Targets of Antimicrobial Agents II. Immunological and Clinical Aspects

The bacterial cell wall represents the primary target for antimicrobial agents. Microbial destruction is accompanied by the release of potent immunostimulatory membrane constituents. Both Gram-positive and Gram-negative bacteria release a variety of lipoproteins and peptidoglycan fragments. Gram-positive bacteria additionally provide lipoteichoic acids, whereas Gram-negative bacteria also release lipopolysaccharide (LPS, endotoxin), essential component of the outer leaflet of the bacterial cell wall and one of the most potent immunostimulatory molecules known. Immune activation therefore can be considered as an adverse effect of antimicrobial destruction and killing during anti-infective treatment. In contrast to antibiotics, the use of cationic amphiphilic antimicrobial peptides allows both effective bacterial killing and inhibition of the immunostimulatory effect of the released bacterial membrane constituents. The administration of antimicrobial peptides alone or in combination with antibiotic agents thus represents a novel strategy in the antiinfective treatment with potentially important beneficial aspects. Here, data are presented which describe immunological and clinical aspects of the use of antimicrobial peptides (AMPs) as therapeutic agents to treat bacterial infection and neutralize the immunostimulatory activity of released cell wall constituents.