Functional analysis of bovine TLR5 and association with IgA responses of cattle following systemic immunisation with H7 flagella

International audienceFlagellin subunits are important inducers of host immune responses through activation of TLR5 when extracellular and the inflammasome if cytosolic. Our previous work demonstrated that systemic immunization of cattle with flagella generates systemic and mucosal IgA responses. The IgA response in mice is TLR5-dependent and TLR5 can impact on the general magnitude of the adaptive response. However, due to sequence differences between bovine and human/murine TLR5 sequences, it is not clear whether bovine TLR5 (bTLR5) is able to stimulate an inflammatory response following interaction with flagellin. To address this we have examined the innate responses of both human and bovine cells containing bTLR5 to H7 flagellin from E. coli O157:H7. Both HEK293 (human origin) and embryonic bovine lung (EBL) cells transfected with bTLR5 responded to addition of H7 flagellin compared to non-transfected controls. Responses were significantly reduced when mutations were introduced into the TLR5-binding regions of H7 flagellin, including an R90T substitution. In bovine primary macrophages, flagellin-stimulated CXCL8 mRNA and secreted protein levels were significantly reduced when TLR5 transcript levels were suppressed by specific siRNAs and stimulation was reduced with the R90T-H7 variant. While these results indicate that the bTLR5 sequence produces a functional flagellin-recognition receptor, cattle immunized with R90T-H7 flagella also demonstrated systemic IgA responses to the flagellin in comparison to adjuvant only controls. This presumably either reflects our findings that R90T-H7 still activates bTLR5, albeit with reduced efficiency compared to WT H7 flagellin, or that other flagellin recognition pathways may play a role in this mucosal response

Systemic Toll-Like Receptor Stimulation Suppresses Experimental Allergic Asthma and Autoimmune Diabetes in NOD Mice

BackgroundInfections 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. Methods and Findings 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)-β and different subsets of regulatory T cells, notably CD4+CD25+FoxP3+ T cells for TLR4 agonists and NKT cells for TLR3 agonists. Conclusions/Significance 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

Intestinal CD103+ dendritic cells: master regulators of tolerance?

CD103+ dendritic cells (DCs) in the intestinal mucosa play a crucial role in tolerance to commensal bacteria and food antigens. These cells originate in the lamina propria (LP) and migrate to the mesenteric lymph nodes (MLNs), where they drive the differentiation of gut-homing FoxP3+ regulatory T cells by producing retinoic acid from dietary vitamin A. Local [`]conditioning' factors in the LP might also contribute to this tolerogenic profile of CD103+ DCs. Considerably less is understood about the generation of active immunity or inflammation in the intestinal mucosa. This might require alterations in pre-existing CD103+ DCs, arrival of new DCs, or the action of a distinct DC population. Here, we discuss our current knowledge of this as yet incompletely understood populatio

The TLR7 agonist R848 alleviates allergic inflammation by targeting invariant NKT cells to produce IFN-gamma

It has been documented that TLR7 stimulation triggers not only antiviral responses, but also alleviates experimental asthma. Considering the implication of invariant NKT (iNKT) cells in both situations, we postulated that they might contribute to the anti-inflammatory effect of TLR7 ligands. We show in this study that spleen cells activated by the TLR7 agonist resiquimod (R848) attenuate allergic inflammation upon adoptive transfer when they are recovered from wild-type, but not from iNKT cell-deficient Jα18(-/-) mice, which proves the specific involvement of this regulatory population. Furthermore, we provide evidence that IFN-γ is critical for the protective effect, which is lost when transferred iNKT cells are sorted from IFN-γ-deficient mice. In support of a direct activation of iNKT cells through TLR7 signaling in vivo, we observed a prompt increase of serum IFN-γ levels, associated with upregulation of CD69 expression on iNKT cells. Moreover, we demonstrate that iNKT cells effectively express TLR7 and respond to R848 in vitro by producing high levels of IFN-γ in the presence of IL-12, consistent with the conclusion that their contribution to the alleviation of allergic inflammation upon treatment with TLR7 ligands is mediated through IFN-γ.status: publishe

The Pro-Th1 cytokine IL-12 enhances IL-4 production by invariant NKT cells: relevance for T cell-mediated hepatitis.

International audienceIL-12 is essential for invariant NKT (iNKT) cells because it can maintain a functionally active population and promote a cytokine profile that is assumed to be mainly of the pro-Th1 type. We used the murine concanavalin A (Con A)-induced hepatitis model, in which iNKT cells, IL-12, IL-4, and IFN-gamma are equally requisite, to reevaluate this issue. We demonstrate that IL-12 interacts directly with iNKT cells, contributes to their recruitment to the liver, and enhances their IL-4 production, which is essential for disease onset. IL-12-deficient mice were less susceptible to experimental hepatitis and their iNKT cells produced less IL-4 than their wild-type counterpart. A normal response could be restored by IL-12 injection, revealing its importance as endogenous mediator. In accordance with this observation, we found that iNKT cells expressed the IL-12R constitutively, in contrast to conventional T cells. Furthermore, the physiological relevance of our data is supported by the lower susceptibility to disease induction of NOD mice, known for their inherent functional and numerical abnormalities of iNKT cells associated with decreased iNKT cell-derived IL-4 production and low IL-12 secretion. Taken together, our findings provide the first evidence that IL-12 can enhance the immune response through increased IL-4 production by iNKT cells, underscoring once more the functional plasticity of this subset

Histological analysis of pancreas.

<p><b>A.</b> Representative photomicrographs of distinct patterns observed in pancreas sections. Histological examination of hematoxylin and eosin stained pancreas sections recovered from the various experimental groups was performed (n = 8 per group). Islet infiltration (insulitis) was scored by deducing the proportion of non-infiltrated islets (healthy islets) and of islets showing a non destructive peripheral insulitis (peri-insulitis) or an invasive/destructive insulitis (destructive insulitis). <b>B.</b> The relative degree of islet inflammation in mice treated with P40, Poly(I∶C), LPS or R848 is shown in a cumulative histogram as compared to PBS-treated controls.</p

Stimulation of TLR pathways prevents spontaneous autoimmune diabetes in NOD mice.

<p><b>A.</b> Female NOD mice were injected <i>i.p.</i> with 200 µg P40, 100 µg Poly(I∶C), 5 µg LPS from <i>S. minnesota</i>, 5 µg Lipid A, 10 µg R848 or PBS once a week, starting at 3–4 weeks of age and for 20 consecutive weeks. Mice were monitored weekly for the advent of glycosuria. Significant prevention from diabetes was observed with most TLR agonists tested (* p<0.05; ** p<0.01) but not with Lipid A. Each panel represents one of 3 independent experiments using 8 mice per group. <b>B.</b> A shorter treatment with the TLR2 agonist P40 administered between 4 and 14 weeks also induced significant protection from diabetes (** p<0.01). <b>C.</b> Treatment with Poly(I∶C) was highly protective if started up to 10 weeks of age but not later (at 16 weeks of age).</p

Stimulation of the TLR/MyD88 pathway modulates immune regulatory cytokines and lymphocyte subsets.

<p><b>A. </b><i>In vitro</i> stimulation of C57BL/6 mouse spleen cells with varying doses of different TLR agonists (P40, 1 or 20 µg/ml; Poly(I∶C), 1 or 10 µg/ml; LPS, 0.1 or 1 µg/ml; R848 0.1 or 1 µg/ml) induced the production of cytokines such as IL-10 (at 48 hrs) and TGF-β (at 72 hrs). Study of splenocytes from MyD88^+/+ or MyD88^−/− C57BL/6 mice confirmed that the effect is dependent on the MyD88 pathway. Results are expressed as mean cytokine level ± SD. Results are representative of three independent experiments. <b>B.</b> Circulating levels of IL-10 and TGF-β were detected following <i>in vivo</i> administration of TLR agonists. NOD mice were injected <i>i.p.</i> with 20 µg of P40, 100 µg of Poly(I∶C) or 10 µg of R848 (n = 8 per group). Control mice were injected with saline (PBS). Sera were collected 24 hours after the injection and cytokine levels were measured by ELISA (*p<0.05; ** p<0.01; *** p<0.005). <b>C.</b> The same conventional treatment protocol (described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011484#pone-0011484-g003" target="_blank">Figure 3</a>) with the TLR4 agonist LPS (5 µg/week/mouse) and the TLR3 agonist Poly(I∶C), that protected wild type NOD mice from diabetes, was applied to female NOD mice invalidated for CD28 (CD28^−/−), CD1d (CD1d^−/−) and IL-4 (IL-4^−/−). Results obtained showed that the Poly(I∶C)-induced protective effect was maintained in CD28^−/− NOD mice (*p<0.05) but not in CD1d^−/− and IL-4^−/− NOD mice. As a mirror-like image the LPS-induced protective effect was maintained in CD1d^−/− and IL-4^−/− NOD mice (* p<0.05) but not in CD28^−/− NOD mice. One representative experiment out of 2 performed is shown.</p