Infections may be associated with exacerbation of allergic and autoimmune diseases. Paradoxically, epidemiological and experimental data have shown that some microorganisms can also prevent these ...pathologies. This observation is at the origin of the hygiene hypothesis according to which the decline of infections in western countries is at the origin of the increased incidence of both Th1-mediated autoimmune diseases and Th2-mediated allergic diseases over the last decades. We have tested whether Toll-like receptor (TLR) stimulation can recapitulate the protective effect of infectious agents on allergy and autoimmunity.
Here, we performed a systematic study of the disease-modifying effects of a set of natural or synthetic TLR agonists using two experimental models, ovalbumin (OVA)-induced asthma and spontaneous autoimmune diabetes, presenting the same genetic background of the non obese diabetic mouse (NOD) that is highly susceptible to both pathologies. In the same models, we also investigated the effect of probiotics. Additionally, we examined the effect of the genetic invalidation of MyD88 on the development of allergic asthma and spontaneous diabetes. We demonstrate that multiple TLR agonists prevent from both allergy and autoimmunity when administered parenterally. Probiotics which stimulate TLRs also protect from these two diseases. The physiological relevance of these findings is further suggested by the major acceleration of OVA-induced asthma in MyD88 invalidated mice. Our results strongly indicate that the TLR-mediated effects involve immunoregulatory cytokines such as interleukin (IL)-10 and transforming growth factor (TGF)-beta and different subsets of regulatory T cells, notably CD4+CD25+FoxP3+ T cells for TLR4 agonists and NKT cells for TLR3 agonists.
These observations demonstrate that systemic administration of TLR ligands can suppress both allergic and autoimmune responses. They provide a plausible explanation for the hygiene hypothesis. They also open new therapeutic perspectives for the prevention of these pathologies.
Outer membrane protein A (OmpA) is a conserved major component of the outer membrane of
Enterobacteriaceae. Here, we report that OmpA from
Klebsiella pneumoniae (KpOmpA) activates macrophages and ...dendritic cells (DCs) in a TLR2-dependent way. However, TLR2 does not account for binding of KpOmpA to innate immune cells. KpOmpA binds the scavenger receptors (SRs) LOX-1 and SREC-I, but not other members of the same family. LOX-1 colocalizes and cooperates with TLR2 in triggering cellular responses. The TLR2-activated functional program includes production of the long pentraxin PTX3, a soluble pattern recognition receptor involved in resistance against diverse pathogens. PTX3, in turn, binds KpOmpA but does not affect recognition of this microbial moiety by cellular receptors. KpOmpA-elicited in vivo inflammation is abrogated in TLR2
−/− mice and significantly reduced in PTX3
−/− mice. Thus, SR-mediated KpOmpA recognition and TLR2-dependent cellular activation set in motion a nonredundant PTX3-mediated humoral amplification loop of innate immunity.
Proteinase 3 (PR3) is the autoantigen in granulomatosis with polyangiitis, an autoimmune necrotizing vasculitis associated with anti-neutrophil cytoplasmic antibodies (ANCAs). Moreover, PR3 is a ...serine protease whose membrane expression can potentiate inflammatory diseases such as ANCA-associated vasculitis and rheumatoid arthritis. During apoptosis, PR3 is co-externalized with phosphatidylserine (PS) and is known to modulate the clearance of apoptotic cells through a calreticulin (CRT)-dependent mechanism. The complement protein C1q is one mediator of efferocytosis, the clearance of altered self-cells, particularly apoptotic cells. Since PR3 and C1q are both involved in the clearance of apoptotic cells and immune response modulation and share certain common ligands (i.e., CRT and PS), we examined their possible interaction. We demonstrated that C1q binding was increased on apoptotic rat basophilic leukemia (RBL) cells that expressed PR3, and we demonstrated the direct interaction between purified C1q and PR3 molecules as shown by surface plasmon resonance. To better understand the functional consequence of this partnership, we tested C1q-dependent phagocytosis of the RBL cell line expressing PR3 and showed that PR3 impaired C1q enhancement of apoptotic cell uptake. These findings shed new light on the respective roles of C1q and PR3 in the elimination of apoptotic cells and suggest a novel potential axis to explore in autoimmune diseases characterized by a defect in apoptotic cell clearance and in the resolution of inflammation.
Proteinase 3 (PR3) is a myeloid serine protease expressed in neutrophils, monocytes, and macrophages. PR3 has a number of well-characterized proinflammatory functions, including cleaving and ...activating chemokines and controlling cell survival and proliferation. When presented on the surface of apoptotic neutrophils, PR3 can disrupt the normal anti-inflammatory reprogramming of macrophages following the phagocytosis of apoptotic cells. To better understand the function of PR3 in vivo, we generated a human PR3 transgenic mouse (hPR3Tg). During zymosan-induced peritonitis, hPR3Tg displayed an increased accumulation of neutrophils within the peritoneal cavity compared with wild-type control mice, with no difference in the recruitment of macrophages or B or T lymphocytes. Mice were also subjected to cecum ligation and puncture, a model used to induce peritoneal inflammation through infection. hPR3Tg displayed decreased survival rates in acute sepsis, associated with increased neutrophil extravasation. The decreased survival and increased neutrophil accumulation were associated with the cleavage of annexin A1, a powerful anti-inflammatory protein known to facilitate the resolution of inflammation. Additionally, neutrophils from hPR3Tg displayed enhanced survival during apoptosis compared with controls, and this may also contribute to the increased accumulation observed during the later stages of inflammation. Taken together, our data suggest that human PR3 plays a proinflammatory role during acute inflammatory responses by affecting neutrophil accumulation, survival, and the resolution of inflammation.
In the present report we have analyzed whether human normal cord blood‐derived mast cells (CBMC) could interact with bacterial products, especially lipopolysaccharide (LPS) from Escherichia coli and ...peptidoglycan (PGN) from Staphylococcus aureus, known as Toll‐like receptor (TLR) 4 and TLR2 agonists, respectively. We found that both LPS and PGN induced significant release of not only tumor necrosis factor‐α (TNF‐α), but also IL‐5, IL‐10 and IL‐13 by human mast cells (MC). We also established that the stimulation of CBMC with LPS or with PGN is mediated through interactions with TLR4 or with TLR2, respectively. Thus, our data indicate that activation of either TLR2 or TLR4 pathway may lead to a pro‐Th2 immune response. However, the release of TNF‐α induced by LPS, conversely to PGN, required the priming of CBMC by IL‐4 and the presence of serum components, in particular soluble CD14. Of interest, stimulation by PGN, but not by LPS, induced release of histamine by human MC. Altogether, these findings provide the first evidence that human MC differentially respond towards bacterial components, and that their responses depend on TLR pathways and reveal human specificities in the pattern of cytokine production.
Stimulation of dendritic cells (DCs) by the egg stage of the helminth parasite Schistosoma mansoni activates a signaling pathway resulting in type I interferon (IFN) and IFN-stimulated gene (ISG) ...expression. Here, we demonstrate that S. mansoni eggs disjointedly activate myeloid differentiation factor 88 (MyD88)-dependent and MyD88-independent pathways in DCs. Inflammatory cytokine expression and NF-κB activation in DCs from MyD88-deficient mice were impaired, whereas signaling transducer activator of transcription (STAT) 1(Tyr701) phosphorylation and ISG expression were intact in MyD88 or Toll-like receptor (TLR)4-deficient counterparts. Accordingly, we analyzed distinct TLR members for their ability to respond to schistosome eggs and established that TLR3 resulted in the activation of NF-κB and the positive regulatory domain III-I site from IFN-β promoter. Unexpectedly, egg-derived RNA possessed RNase A-resistant and RNase III-sensitive structures capable of triggering TLR3 activation, suggesting the involvement of double-stranded (ds) structures. Moreover, DCs from TLR3-deficient mice displayed a complete loss of signaling transducer activator of transcription 1 phosphorylation and ISG expression in response to egg-derived dsRNA. Finally, TLR3-deficient DCs showed a reduced response to schistosome eggs relative to wild-type cells. Collectively, our data suggest for the first time that dsRNA from a non-viral pathogen may act as an inducer of the innate immune system through TLR3.
Transforming Growth Factor-β and Natural Killer T-Cells Are Involved in the Protective Effect of a Bacterial Extract on Type
1 Diabetes
Marie-Alexandra Alyanakian 1 ,
Françoise Grela 2 ,
Aude ...Aumeunier 2 ,
Carlo Chiavaroli 3 ,
Christine Gouarin 1 ,
Emilie Bardel 2 ,
Gérard Normier 3 ,
Lucienne Chatenoud 1 ,
Nathalie Thieblemont 2 and
Jean-François Bach 1
1 Université René Descartes Paris 5, Institut National de la Santé et de la Recherche Médicale U580, Hôpital Necker-Enfants
Malades, Paris, France
2 Université René Descartes Paris 5, Unité Mixte de Recherche (UMR), National Center for Scientific Research (CNRS) 8147, Hôpital
Necker-Enfants Malades, Paris, France
3 OM Pharma, Meyrin, Switzerland
Address correspondence and reprint requests to Jean-François Bach, MD, DSc, INSERM U580, Hôpital Necker-Enfants Malades, 161
Rue de Sèvres, 75015 Paris, France. E-mail: bach{at}necker.fr
Abstract
The onset of type 1 diabetes in NOD mice is delayed by oral administration of a bacterial extract (OM-85) and can be completely
prevented by its intraperitoneal administration. Optimal prevention is observed when starting treatment at 3 or 6 weeks of
age, and some effect is still observed with treatment at 10 weeks of age. Using genetically deficient mice and cytokine-neutralizing
monoclonal antibodies, we demonstrate here that the therapeutic effect does not involve T-helper type 2 cytokines (interleukin
IL-4 and -10) but is tightly dependent on transforming growth factor (TGF)-β. Natural killer T-cells also participate in
the therapeutic effect because CD1d −/− NOD mice are partially resistant to the protective effect of OM-85. The question remains of the specificity of the protective
effect of OM-85, which may include proinflammatory components. It will thus be important to further characterize the molecular
components that afford protection from type 1 diabetes. Lipopolysaccharide is excluded, but other Toll-like receptor (TLR)
agonists could be involved because OM-85 stimulated dendritic cells and induced TGF-β production by splenocytes in a TLR-2–,
TLR-4–, and MyD88-dependent fashion.
CFA, complete Freund’s adjuvant
IFA, incomplete Freund’s adjuvant
IFN-γ, γ-interferon
IL, interleukin
MDP, muramyldipeptide
NKT, natural killer T
TGF, transforming growth factor
TLR, Toll-like receptor
Footnotes
Accepted September 27, 2005.
Received February 14, 2005.
DIABETES
Recent evidence has highlighted the role of histamine in inflammation. Since this monoamine has also been strongly implicated in the pathogenesis of type-1 diabetes, we assessed its effect in the ...nonobese diabetic (NOD) mouse model. To this end, we used mice (inactivated) knocked out for the gene encoding histidine decarboxylase, the unique histamine-forming enzyme, backcrossed on a NOD genetic background. We found that the lack of endogenous histamine in NOD HDC−/− mice decreased the incidence of diabetes in relation to their wild-type counterpart. Whereas the proportion of regulatory T and myeloid-derived suppressive cells was similar in both strains, histamine deficiency was associated with increased levels of immature macrophages, as compared with wild-type NOD mice. Concerning the cytokine pattern, we found a decrease in circulating IL-12 and IFN-γ in HDC−/− mice, while IL-6 or leptin remained unchanged, suggesting that histamine primarily modulates the inflammatory environment. Paradoxically, exogenous histamine given to NOD HDC−/− mice provided also protection against T1D. Our study supports the notion that histamine is involved in the pathogenesis of diabetes, thus providing additional evidence for its role in the regulation of the immune response.
Addition of lipopolysaccharide (LPS) to cells in the form of LPS-soluble (s)CD14 complexes induces strong cellular responses. During this process, LPS is delivered from sCD14 to the plasma membrane, ...and the cell-associated LPS is then rapidly transported to an intracellular site. This transport appears to be important for certain cellular responses to LPS, as drugs that block transport also inhibit signaling and cells from LPS-hyporesponsive C3H/HeJ mice fail to exhibit this transport. To identify the intracellular destination of fluorescently labeled LPS after its delivery from sCD14 into cells, we have made simultaneous observations of different organelles using fluorescent vital dyes or probes. Endosomes, lysosomes, the endoplasmic reticulum, and the Golgi apparatus were labeled using Texas red (TR)-dextran, LysoTrackertrade mark Red DND-99, DiOC6(3), and boron dipyrromethane (BODIPY)-ceramide, respectively. After 30 min, LPS did not colocalize with endosomes, lysosomes, or endoplasmic reticulum in polymorphonuclear leukocytes, although some LPS-positive vesicles overlapped with the endosomal marker, fluorescent dextran. On the other hand, LPS did appear to colocalize with two markers of the Golgi apparatus, BODIPY-ceramide and TRITC (tetramethylrhodamine isothiocyanate)-labeled cholera toxin B subunit. We further confirmed the localization of LPS in the Golgi apparatus using an epithelial cell line, HeLa, which responds to LPS-sCD14 complexes in a CD14-dependent fashion: BODIPY-LPS was internalized and colocalized with fluorescently labeled Golgi apparatus probes in live HeLa cells. Morphological disruption of the Golgi apparatus in brefeldin A-treated HeLa cells caused intracellular redistribution of fluorescent LPS. These results are consistent with the Golgi apparatus being the primary delivery site of monomeric LPS.
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