Alexandre Castro Keller

Identification of an IL-17–producing NK1.1neg iNKT cell population involved in airway neutrophilia

Invariant natural killer T (iNKT) cells are an important source of both T helper type 1 (Th1) and Th2 cytokines, through which they can exert beneficial, as well as deleterious, effects in a variety of inflammatory diseases. This functional heterogeneity raises the question of how far phenotypically distinct subpopulations are responsible for such contrasting activities. In this study, we identify a particular set of iNKT cells that lack the NK1.1 marker (NK1.1neg) and secrete high amounts of interleukin (IL)-17 and low levels of interferon (IFN)-γ and IL-4. NK1.1neg iNKT cells produce IL-17 upon synthetic (α-galactosylceramide [α-GalCer] or PBS-57), as well as natural (lipopolysaccharides or glycolipids derived from Sphingomonas wittichii and Borrelia burgdorferi), ligand stimulation. NK1.1neg iNKT cells are more frequent in the lung, which is consistent with a role in the natural immunity to inhaled antigens. Indeed, airway neutrophilia induced by α-GalCer or lipopolysaccharide instillation was significantly reduced in iNKT-cell–deficient Jα18−/− mice, which produced significantly less IL-17 in their bronchoalveolar lavage fluid than wild-type controls. Furthermore, airway neutrophilia was abolished by a single treatment with neutralizing monoclonal antibody against IL-17 before α-GalCer administration. Collectively, our findings reveal that NK1.1neg iNKT lymphocytes represent a new population of IL-17–producing cells that can contribute to neutrophil recruitment through preferential IL-17 secretion.

Cutting Edge: Invariant Vα14 NKT Cells Are Required for Allergen-Induced Airway Inflammation and Hyperreactivity in an Experimental Asthma Model

Airway hyperreactivity (AHR), eosinophilic inflammation with a Th2-type cytokine profile, and specific Th2-mediated IgE production characterize allergic asthma. In this paper, we show that OVA-immunized Jα18−/− mice, which are exclusively deficient in the invariant Vα14+ (iVα14), CD1d-restricted NKT cells, exhibit impaired AHR and airway eosinophilia, decreased IL-4 and IL-5 production in bronchoalveolar lavage fluid, and reduced OVA-specific IgE compared with wild-type (WT) littermates. Adoptive transfer of WT iVα14 NKT cells fully reconstitutes the capacity of Jα18−/− mice to develop allergic asthma. Also, specific tetramer staining shows that OVA-immunized WT mice have activated (CD69+) iVα14 NKT cells. Importantly, anti-CD1d mAb treatment blocked the ability of iVα14 T cells to amplify eosinophil recruitment to airways, and both Th2 cytokine and IgE production following OVA challenge. In conclusion, these findings clearly demonstrate that iVα14 NKT cells are required to participate in allergen-induced Th2 airway inflammation through a CD1d-dependent mechanism.

Critical role of ROR-γt in a new thymic pathway leading to IL-17-producing invariant NKT cell differentiation

Invariant natural killer T (iNKT) cells constitute a subpopulation of T cells that recognize glycolipids presented by CD1d molecules. They are characterized by their prompt production of interleukin-4 (IL-4) and interferon-γ (IFN-γ), which enables them to modulate diverse immune responses. Recently, we enlarged this concept by identifying a distinct IL-17-producing iNKT cell subset, named iNKT17 cells. The mechanisms leading to the acquisition of this new iNKT cell activity are unknown. Herein we show that IL-17-producing iNKT cells are already present in the thymus, predominantly among a subset regarded so far as an immature stage of thymic iNKT cell development, the CD1d tetramerposCD44posNK1.1negCD4neg cells. Using EGFP reporter mice, we demonstrate that the transcription factor ROR-γt is critical for the thymic differentiation of this subset because only ROR-γtpos iNKT cells are capable of massively secreting IL-17. Moreover, IL-17-producing CD1d tetramerposCD44posNK1.1negCD4neg thymic iNKT cells have reached a mature differentiation stage because they fail to generate other cell subsets in fetal thymic organ culture. Conversely, thymic ROR-γtneg iNKT cell precursors give rise to progeny, but acquire neither ROR-γt expression nor the ability to secrete IL-17. In conclusion, our findings demonstrate an alternative thymic pathway leading to the development of iNKT17 cells that requires ROR-γt expression.

Bacterial Lipopolysaccharide Signaling Through Toll-Like Receptor 4 Suppresses Asthma-Like Responses Via Nitric Oxide Synthase 2 Activity

Asthma results from an intrapulmonary allergen-driven Th2 response and is characterized by intermittent airway obstruction, airway hyperreactivity, and airway inflammation. An inverse association between allergic asthma and microbial infections has been observed. Microbial infections could prevent allergic responses by inducing the secretion of the type 1 cytokines, IL-12 and IFN-γ. In this study, we examined whether administration of bacterial LPS, a prototypic bacterial product that activates innate immune cells via the Toll-like receptor 4 (TLR4) could suppress early and late allergic responses in a murine model of asthma. We report that LPS administration suppresses the IgE-mediated and mast cell-dependent passive cutaneous anaphylaxis, pulmonary inflammation, airway eosinophilia, mucus production, and airway hyperactivity. The suppression of asthma-like responses was not due to Th1 shift as it persisted in IL-12−/− or IFN-γ−/− mice. However, the suppressive effect of LPS was not observed in TLR4- or NO synthase 2-deficient mice. Our findings demonstrate, for the first time, that LPS suppresses Th2 responses in vivo via the TLR4-dependent pathway that triggers NO synthase 2 activity.

Toll-like receptor 4 agonists adsorbed to aluminium hydroxide adjuvant attenuate ovalbumin-specific allergic airway disease: role of MyD88 adaptor molecule and interleukin-12/interferon-gamma axis.

Background Epidemiological and experimental data suggest that bacterial lipopolysaccharides (LPS) can either protect from or exacerbate allergic asthma. Lipopolysaccharides trigger immune responses through toll‐like receptor 4 (TLR4) that in turn activates two major signalling pathways via either MyD88 or TRIF adaptor proteins. The LPS is a pro‐Type 1 T helper cells (Th1) adjuvant while aluminium hydroxide (alum) is a strong Type 2 T helper cells (Th2) adjuvant, but the effect of the mixing of both adjuvants on the development of lung allergy has not been investigated.

Invariant Natural Killer T Cells and TGF-β Attenuate Anti-GBM Glomerulonephritis

Invariant natural killer T (iNKT) cells represent a particular subset of T lymphocytes capable of producing several cytokines, which exert regulatory or effector functions, following stimulation of the T cell receptor. In this study, we investigated the influence of iNKT cells on the development of experimental anti-glomerular basement membrane glomerulonephritis (anti-GBM GN). After injection of anti-GBM serum, the number of kidney iNKT cells rapidly increased. iNKT cell-deficient mice (Jalpha18-/-) injected with anti-GBM serum demonstrated worse renal function, increased proteinuria, and greater glomerular and tubular injury compared with similarly treated wild-type mice. We did not detect significant differences in Th1/Th2 polarization in renal tissue that might have explained the severity of disease in Jalpha18-/- mice. Interestingly, expression of both TGF-beta and TGF-beta-induced (TGFBI) mRNA was higher in wild-type kidneys compared with Jalpha18-/- kidneys, suggesting a possible protective role for TGF-beta in anti-GBM GN. Administration of an anti-TGF-beta neutralizing antibody significantly enhanced the severity of disease in wild-type, but not Jalpha18-/-, mice. In conclusion, in experimental anti-GBM GN, iNKT cells attenuate disease severity and TGF-beta has a renoprotective role.

Regulatory T Cells Accumulate in the Lung Allergic Inflammation and Efficiently Suppress T-Cell Proliferation but Not Th2 Cytokine Production

Foxp3+CD25+CD4+ regulatory T cells are vital for peripheral tolerance and control of tissue inflammation. In this study, we characterized the phenotype and monitored the migration and activity of regulatory T cells present in the airways of allergic or tolerant mice after allergen challenge. To induce lung allergic inflammation, mice were sensitized twice with ovalbumin/aluminum hydroxide gel and challenged twice with intranasal ovalbumin. Tolerance was induced by oral administration of ovalbumin for 5 consecutive days prior to OVA sensitization and challenge. We detected regulatory T cells (Foxp3+CD25+CD4+ T cells) in the airways of allergic and tolerant mice; however, the number of regulatory T cells was more than 40-fold higher in allergic mice than in tolerant mice. Lung regulatory T cells expressed an effector/memory phenotype (CCR4highCD62LlowCD44highCD54highCD69+) that distinguished them from naive regulatory T cells (CCR4intCD62LhighCD44intCD54intCD69−). These regulatory T cells efficiently suppressed pulmonary T-cell proliferation but not Th2 cytokine production.

Nitric oxide paradox in asthma

Asthma results from allergen-driven intrapulmonary Th2 response, and is characterized by intermittent airway obstruction, airway hyperreactivity (AHR), and airway inflammation. Accumulating evidence indicates that inflammatory diseases of the respiratory tract are commonly associated with elevated production of nitric oxide (NO). It has been shown that exhaled NO may be derived from constitutive NO synthase (NOS) such as endothelial (NOS 3) and neural (NOS 1) in normal airways, while increased levels of NO in asthma appear to be derived from inducible NOS2 expressed in the inflamed airways. Nevertheless, the functional role of NO and NOS isoforms in the regulation of AHR and airway inflammation in human or experimental models of asthma is still highly controversial. In the present commentary we will discuss the role of lipopolysaccharides contamination of allergens as key element in the controversy related to the regulation of NOS2 activity in experimental asthma.

Unconventional Strategies for the Suppression of Allergic Asthma

Allergic asthma results from an intrapulmonary allergen-driven Th2 response and is characterized by intermittent airway obstruction, airway hyperreactivity, and airway inflammation. An inverse association between allergic asthma and microbial infections has been observed. And this observation constitutes the base of the hygiene hypothesis. Here we discuss the hygiene hypothesis with emphasis on regulatory cells. We review the evidence for the emergence of regulatory cells, such as CD4(+)CD25(+) T cells during infection or during induction of tolerance by mucosal antigen administration. The review focuses also on the emergence of activated CD8(+) T cells and macrophages, induced by infections or microbial products, which also can result in the suppression of asthma. The underlying mechanisms by which regulatory immune cells suppress asthma may represent novel unconventional strategies controlling asthma.

Decreased nasal tolerance to allergic asthma in mice fed an amino acid-based protein-free diet.

Abstract: Intranasal (i.n.) administration of soluble proteins induces a state of specific unresponsiveness to subsequent immunization, known as nasal tolerance. It is thought that newborns are less susceptible to nasal tolerance induction. Recently, we have shown that feeding adult animals with a protein‐free diet (Aa) resulted in their arrest at an immature immunological profile. Here, we examined the effects of the Aa diet on the development of nasal tolerance to ovalbumin (OVA) in a murine model of allergic asthma. Nasal OVA administration suppressed almost totally the OVA‐induced asthma‐like responses (airway eosinophilia, type 2 cytokine production, and OVA‐specific IgE antibodies) in chow‐ or casein‐fed BALB/c mice. In contrast, in Aa‐fed animals the suppression of asthma‐like responses by nasal OVA was partial, being effective in suppressing airway eosinophilia, but not airway type 2 cytokine or OVA‐specific IgE response. We conclude that animals fed the Aa diet are more resistant to the induction of nasal tolerance. Our animal model may mimic the features of the immune system of human infants.