Catherine Werts

Lymphoid tissue genesis induced by commensals through NOD1 regulates intestinal homeostasis.

Intestinal homeostasis is critical for efficient energy extraction from food and protection from pathogens. Its disruption can lead to an array of severe illnesses with major impacts on public health, such as inflammatory bowel disease characterized by self-destructive intestinal immunity. However, the mechanisms regulating the equilibrium between the large bacterial flora and the immune system remain unclear. Intestinal lymphoid tissues generate flora-reactive IgA-producing B cells, and include Peyers patches and mesenteric lymph nodes, as well as numerous isolated lymphoid follicles (ILFs). Here we show that peptidoglycan from Gram-negative bacteria is necessary and sufficient to induce the genesis of ILFs in mice through recognition by the NOD1 (nucleotide-binding oligomerization domain containing 1) innate receptor in epithelial cells, and β-defensin 3- and CCL20-mediated signalling through the chemokine receptor CCR6. Maturation of ILFs into large B-cell clusters requires subsequent detection of bacteria by toll-like receptors. In the absence of ILFs, the composition of the intestinal bacterial community is profoundly altered. Our results demonstrate that intestinal bacterial commensals and the immune system communicate through an innate detection system to generate adaptive lymphoid tissues and maintain intestinal homeostasis.

Leptospiral lipopolysaccharide activates cells through a TLR2-dependent mechanism

Leptospira interrogans are zoonotic pathogens that have been linked to a recent increased incidence of morbidity and mortality in highly populated tropical urban centers. They are unique among invasive spirochetes in that they contain outer membrane lipopolysaccharide (LPS) as well as lipoproteins. Here we show that both these leptospiral outer membrane constituents activate macrophages through CD14 and the Toll-like receptor 2 (TLR2). Conversely, it seems that TLR4, a central component for recognition of Gram-negative LPS, is not involved in cellular responses to L. interrogans. We also show that for intact L. interrogans, it is LPS, not lipoprotein, that constitutes the predominant signaling component for macrophages through a TLR2 pathway. These data provide a basis for understanding the innate immune response caused by leptospirosis and demonstrate a new ligand specificity for TLR2.

Toll-like receptor 2-dependent bacterial sensing does not occur via peptidoglycan recognition

Toll‐like receptor 2 (TLR2) has been shown to recognize several classes of pathogen‐associated molecular patterns including peptidoglycan (PG). However, studies linking PG with TLR2 recognition have relied mainly on the use of commercial Staphylococcus aureus PG and have not addressed TLR2 recognition of other PG types. Using highly purified PGs from eight bacteria (Escherichia coli, Pseudomonas aeruginosa, Yersinia pseudotuberculosis, Helicobacter pylori, Bacillus subtilis, Listeria monocytogenes, Streptococcus pneumoniae and S. aureus), we show that these PGs are not sensed through TLR2, TLR2/1 or TLR2/6. PG sensing is lost after removal of lipoproteins or lipoteichoic acids (LTAs) from Gram‐negative and Gram‐positive cell walls, respectively. Accordingly, purified LTAs are sensed synergistically through TLR2/1. Finally, we show that elicited peritoneal murine macrophages do not produce tumour necrosis factor‐α or interleukin‐6 in response to purified PGs, suggesting that PG detection is more likely to occur intracellularly (through Nod1/Nod2) rather than from the extracellular compartment.

Synergistic stimulation of human monocytes and dendritic cells by Toll-like receptor 4 and NOD1- and NOD2-activating agonists

Muropeptides are degradation products of bacterial peptidoglycan (PG) sensed by nucleotide‐binding oligomerization domain 1 (NOD1) and NOD2, members of a recently discovered family of pattern recognition molecules (PRM). One of these muropeptides, muramyl dipeptide (MDP) mediates cell signaling by NOD2, exerts adjuvant activity and synergizes with lipopolysaccharide (LPS) to induce pro‐inflammatory responses in vitro and in vivo. In contrast, few and contradictory results exist about the stimulatory capacity of NOD1 agonists. Thus, the ability of NOD1 (MurNAc‐L‐Ala‐γ‐D‐Glu‐meso‐diaminopimelic acid, MtriDAP) and NOD2 (MurNAc‐L‐Ala‐D‐isoGln, MDP; MurNAc‐L‐Ala‐γ‐D‐Glu‐L‐Lys, MtriLYS) agonists to activate primary human myeloid cells was examined. We show that both CD14+ monocytes and CD1a+ immature dendritic cells (DC) express NOD1 and NOD2 mRNA. Stimulation of primary human monocytes and DC with highly purified muropeptides (MtriDAP, MDP and MtriLYS) induces release of pro‐inflammatory cytokines. We reveal here that NOD1 as well as NOD2 agonists act cooperatively with LPS to stimulate the release of both pro‐ and anti‐inflammatory cytokines in these myeloid cell subsets. Finally, we report that NOD1 as well as NOD2 agonists synergize with sub‐active doses of LPS to induce DC maturation, demonstrating that NOD agonists act cooperatively with molecules sensed by Toll‐like receptor 4 to instruct the onset of adaptive immune responses.

The Ubiquitin-Editing Enzyme A20 Restricts Nucleotide-Binding Oligomerization Domain Containing 2-Triggered Signals

Muramyl dipeptide (MDP), a product of bacterial cell-wall peptidoglycan, activates innate immune cells by stimulating nucleotide-binding oligomerization domain containing 2 (NOD2) -dependent activation of the transcription factor NFkappaB and transcription of proinflammatory genes. A20 is a ubiquitin-modifying enzyme that restricts tumor necrosis factor (TNF) receptor and Toll-like receptor (TLR) -induced signals. We now show that MDP induces ubiquitylation of receptor- interacting protein 2 (RIP2) in primary macrophages. A20-deficient cells exhibit dramatically amplified responses to MDP, including increased RIP2 ubiquitylation, prolonged NFkappaB signaling, and increased production of proinflammatory cytokines. In addition, in vivo responses to MDP are exaggerated in A20-deficient mice and in chimeric mice bearing A20-deficient hematopoietic cells. These exaggerated responses occur independently of the TLR adaptors MyD88 and TRIF as well as TNF signals. These findings indicate that A20 directly restricts NOD2 induced signals in vitro and in vivo, and provide new insights into how these signals are physiologically restricted.

TIR, CARD and PYRIN : three domains for an antimicrobial triad

Innate immunity to microorganisms in mammals has gained a substantial interest during the last decade. The discovery of the Toll-like receptor (TLR) family has allowed the identification of a class of membrane-spanning receptors dedicated to microbial sensing. TLRs transduce downstream signaling via their intracellular Toll–interleukin-1 receptor (TIR) domain. More recently, the role of intracellular microbial sensors has been uncovered. These molecules include the Nod-like receptors Nod1, Nod2, Ipaf and Nalps, together with the helicase domain-containing antiviral proteins RIG-I and Mda-5. The intracellular microbial sensors lack the TIR domain, but instead transduce downstream signals via two domains also implicated in homophilic protein–protein interactions, the caspase activation and recruitment domain (CARD) and PYRIN domains. In light with these recent findings, we propose that TIR, CARD and PYRIN domains represent the three arms of innate immune detection of microorganisms in mammals.

Differential TLR recognition of leptospiral lipid A and lipopolysaccharide in murine and human cells.

Leptospira interrogans is a spirochete that is responsible for leptospirosis, a zoonotic disease. This bacterium possesses an unusual LPS that has been shown to use TLR2 instead of TLR4 for signaling in human cells. The structure of its lipid A was recently deciphered. Although its overall hexa-acylated disaccharide backbone is a classical feature of all lipid A forms, the lipid A of L. interrogans is peculiar. In this article, the functional characterization of this lipid A was studied in comparison to whole parental leptospiral LPS in terms of cell activation and use of TLR in murine and human cells. Lipid A from L. interrogans did not coagulate the Limulus hemolymph. Although leptospiral lipid A activated strongly murine RAW cells, it did not activate human monocytic cells. Results obtained from stimulation of peritoneal-elicited macrophages from genetically deficient mice for TLR2 or TLR4 clearly showed that lipid A stimulated the cells through TLR4 recognition, whereas highly purified leptospiral LPS utilized TLR2 as well as TLR4. In vitro experiments with transfected human HEK293 cells confirmed that activation by lipid A occurred only through murine TLR4-MD2 but not through human TLR4-MD2, nor murine or human TLR2. Similar studies with parental leptospiral LPS showed that TLR2/TLR1 were the predominant receptors in human cells, whereas TLR2 but also TLR4 contributed to activation in murine cells. Altogether these results highlight important differences between human and mouse specificity in terms of TLR4-MD2 recognition that may have important consequences for leptospiral LPS sensing and subsequent susceptibility to leptospirosis.

Role of AmiA in the morphological transition of Helicobacter pylori and in immune escape.

The human gastric pathogen Helicobacter pylori is responsible for peptic ulcers and neoplasia. Both in vitro and in the human stomach it can be found in two forms, the bacillary and coccoid forms. The molecular mechanisms of the morphological transition between these two forms and the role of coccoids remain largely unknown. The peptidoglycan (PG) layer is a major determinant of bacterial cell shape, and therefore we studied H. pylori PG structure during the morphological transition. The transition correlated with an accumulation of the N-acetyl-D-glucosaminyl-β(1,4)-N-acetylmuramyl-L-Ala–D-Glu (GM-dipeptide) motif. We investigated the molecular mechanisms responsible for the GM-dipeptide motif accumulation, and studied the role of various putative PG hydrolases in this process. Interestingly, a mutant strain with a mutation in the amiA gene, encoding a putative PG hydrolase, was impaired in accumulating the GM-dipeptide motif and transforming into coccoids. We investigated the role of the morphological transition and the PG modification in the biology of H. pylori. PG modification and transformation of H. pylori was accompanied by an escape from detection by human Nod1 and the absence of NF-κB activation in epithelial cells. Accordingly, coccoids were unable to induce IL-8 secretion by AGS gastric epithelial cells. amiA is, to our knowledge, the first genetic determinant discovered to be required for this morphological transition into the coccoid forms, and therefore contributes to modulation of the host response and participates in the chronicity of H. pylori infection.

Nod1 and Nod2 induce CCL5/RANTES through the NF‐κB pathway

The Nod‐like receptor proteins Nod1 and Nod2 participate in innate immune responses against bacteria through intracellular detection of peptidoglycan, a component of bacterial cell wall. Recent evidence has demonstrated that Nod1 stimulates the release of chemokines that attract neutrophils at the site of infection, such as CXCL8/IL‐8 in humans, and CXCL1/keratinocyte‐derived chemokine and CXCL2/MIP‐2 in mice. We aimed to determine whether Nod proteins could trigger the release of CCL5/RANTES, a chemokine known to attract a number of immune cells, but not neutrophils. Our results demonstrate that activation of both Nod1 and Nod2 results in substantial secretion of CCL5 by murine macrophages. Moreover, in vivo, the intraperitoneal injection of murine Nod1 or Nod2 agonists resulted in a rapid secretion of CCL5 into the bloodstream. We also observed that Nod‐dependent secretion of CCL5 did not correlate with the induction of the interferon‐β pathway, a major signaling cascade for the activation of CCL5 by viruses. In contrast, we identified a key role of the NF‐κB pathway in Nod‐dependent stimulation of the CCL5 promoter. Together, these results identify a novel target downstream of Nod1 and Nod2, which is likely to play a key role in orchestrating the global Nod‐dependent immune defense during bacterial infections.

TLR4- and TLR2-Mediated B Cell Responses Control the Clearance of the Bacterial Pathogen, Leptospira interrogans

Leptospirosis is a widespread zoonosis caused by pathogenic Leptospira interrogans that are transmitted by asymptomatic infected rodents. Leptospiral lipoproteins and LPS have been shown to stimulate murine cells via TLRs 2 and 4. Host defense mechanisms remain obscure, although TLR4 has been shown to be involved in clearing Leptospira. In this study, we show that double (TLR2 and TLR4) knockout (DKO) mice rapidly died from severe hepatic and renal failure following Leptospira inoculation. Strikingly, the severe proinflammatory response detected in the liver and kidney from Leptospira-infected DKO mice appears to be independent of MyD88, the main adaptor of TLRs. Infection of chimeric mice constructed with wild-type and DKO mice, and infection of several lines of transgenic mice devoid of T and/or B lymphocytes, identified B cells as the crucial lymphocyte subset responsible for the clearance of Leptospira, through the early production of specific TLR4-dependent anti-Leptospira IgMs elicited against the leptospiral LPS. We also found a protective tissue compartmentalized TLR2/TLR4-mediated production of IFN-γ by B and T lymphocytes, in the liver and kidney, respectively. In contrast, the tissue inflammation observed in Leptospira-infected DKO mice was further characterized to be mostly due to B lymphocytes in the liver and T cells in the kidney. Altogether these findings demonstrate that TLR2 and TLR4 play a key role in the early control of leptospirosis, but do not directly trigger the inflammation induced by pathogenic Leptospira.