Luciana Mirotti

Regulatory T Cells Migrate to Airways via CCR4 and Attenuate the Severity of Airway Allergic Inflammation

We have previously shown that regulatory T (Treg) cells that accumulate in the airways of allergic mice upregulate CC-chemokine receptor 4 (CCR4) expression. These Treg cells suppressed in vitro Th2 cell proliferation but not type 2 cytokine production. In the current study, using a well-established murine model of allergic lung disease or oral tolerance, we evaluated the in vivo activity of Treg cells in allergic airway inflammation with special focus on CCR4 function. We found that allergic, but not tolerant, mice treated with anti-CD25 Ab showed increased airway eosinophilia and IL-5– or IL-4–producing Th2 cells when compared with untreated mice. Notably, mice with CCR4 deficiency displayed an augmented airway allergic inflammation compared with wild-type or CCR2 knockout (KO) mice. The allergic phenotype of CCR4KO mice was similar to that observed in anti-CD25–treated mice. The exacerbated allergic inflammation of CCR4KO mice was directly associated with an impaired migration of Treg cells to airways and augmented frequency of pulmonary Th2 cells. Adoptive transfer of CD25+CD4+ T cells expressing high levels of CCR4, but not CCR4KO CD25+CD4+ T cells, attenuated the severe airway Th2 response of CCR4KO mice. Our results show that CCR4 is critically involved in the migration of Treg cells to allergic lungs that, in turn, attenuate airway Th2 activation and allergic eosinophilic inflammation.

Lipids are required for the development of Brazil nut allergy: the role of mouse and human iNKT cells

Lipids are required for mice sensitization to Ber e 1, Brazil nut major allergen. Here, we characterized different lipid fractions extracted from Brazil nuts and the lipid‐binding ability of Ber e 1. Further, we determined their in vivo ability to induce Ber‐specific anaphylactic antibodies and the role of invariant natural killer T (iNKT) cells in this process.

Endotoxin Exposure during Sensitization to Blomia tropicalis Allergens Shifts TH2 Immunity Towards a TH17-Mediated Airway Neutrophilic Inflammation: Role of TLR4 and TLR2

Experimental evidence and epidemiological studies indicate that exposure to endotoxin lipopolysaccharide (eLPS) or other TLR agonists prevent asthma. We have previously shown in the OVA-model of asthma that eLPS administration during alum-based allergen sensitization blocked the development of lung TH2 immune responses via MyD88 pathway and IL-12/IFN-γ axis. In the present work we determined the effect of eLPS exposure during sensitization to a natural airborne allergen extract derived from the house dust mite Blomia tropicalis (Bt). Mice were subcutaneously sensitized with Bt allergens co-adsorbed onto alum with or without eLPS and challenged twice intranasally with Bt. Cellular and molecular parameters of allergic lung inflammation were evaluated 24 h after the last Bt challenge. Exposure to eLPS but not to ultrapure LPS (upLPS) preparation during sensitization to Bt allergens decreased the influx of eosinophils and increased the influx of neutrophils to the airways. Inhibition of airway eosinophilia was not observed in IFN-γdeficient mice while airway neutrophilia was not observed in IL-17RA-deficient mice as well in mice lacking MyD88, CD14, TLR4 and, surprisingly, TLR2 molecules. Notably, exposure to a synthetic TLR2 agonist (PamCSK4) also induced airway neutrophilia that was dependent on TLR2 and TLR4 molecules. In the OVA model, exposure to eLPS or PamCSK4 suppressed OVA-induced airway inflammation. Our results suggest that B. tropicalis allergens engage TLR4 that potentiates TLR2 signaling. This dual TLR activation during sensitization results in airway neutrophilic inflammation associated with increased frequency of lung TH17 cells. Our work highlight the complex interplay between bacterial products, house dust mite allergens and TLR signaling in the induction of different phenotypes of airway inflammation.

Adsorption of Toll-Like Receptor 4 Agonist to Alum-Based Tetanus Toxoid Vaccine Dampens Pro-T Helper 2 Activities and Enhances Antibody Responses.

Aluminum salts gels (alum) are TLR-independent adjuvants and have been used to boost antibody responses in alum-based vaccines such as diphtheria, pertussis, and tetanus toxoid (DPT) triple vaccine. However, the pro-Th2 activity of alum-based vaccine formulations has not been fully appreciated. Here we found that alum-based tetanus toxoid (TT) vaccine was biased toward a Th-2 profile as shown by TT-induced airway eosinophilic inflammation, type 2 cytokine production, and high levels of IgE anaphylactic antibodies. The adsorption into alum of prototypic TLR4 agonists such as lipopolysaccharides (LPS) derived from Escherichia coli consistently dampened TT-induced Th2 activities without inducing IFNγ or Th1-like responses in the lung. Conversely, adsorption of monophosphoryl lipid A (MPLA) extracted from Salmonella minnesota, which is a TIR-domain-containing adapter-inducing interferon-β- (TRIF-) biased TLR4 agonist, was less effective in decreasing Th-2 responses. Importantly, in a situation with antigenic competition (OVA plus TT), TT-specific IgG1 or IgG2a was decreased compared with TT sensitization. Notably, LPS increased the production of IgG1 and IgG2a TT-specific antibodies. In conclusion, the addition of LPS induces a more robust IgG1 and IgG2a TT-specific antibody production and concomitantly decreases Th2-cellular and humoral responses, indicating a potential use of alum/TLR-based vaccines.

Neural pathways in allergic inflammation.

Allergy is on the rise worldwide. Asthma, food allergy, dermatitis, and systemic anaphylaxis are amongst the most common allergic diseases. The association between allergy and altered behavior patterns has long been recognized. The molecular and cellular pathways in the bidirectional interactions of nervous and immune systems are now starting to be elucidated. In this paper, we outline the consequences of allergic diseases, especially food allergy and asthma, on behavior and neural activity and on the neural modulation of allergic responses.

Food aversion: A critical balance between allergen-specific IgE levels and taste preference

Animals sensitized to allergens change their feeding behavior and avoid drinking the otherwise preferred sweetened solutions containing the allergens. This phenomenon, known as food aversion, appears to be mediated by allergen-specific IgE antibodies. Here we investigated food aversion in BALB/c and C57BL/6 mice, which differ in their allergic responses to the allergen ovalbumin as well as in their preference for sweet taste. BALB/c mice present higher levels of IgE and a natural lower preference for sweet flavors when compared to C57BL/6 mice. Specifically, we studied a conflicting situation in which animals simultaneously experienced the aversive contact with the allergen and the attractive sweet taste of increasing concentrations of sucrose. We found that BALB/c mice were more prone to develop food aversion than C57BL/6 mice and that this aversive behavior could be abolished in both strains by increasing the palatability of the solution containing the allergen. In both strains food aversion was positively correlated with the levels of allergen-specific IgE antibodies and inversely correlated with their preference for sucrose sweetened solutions.

Role of m2 muscarinic receptor in the airway response to methacholine of mice selected for minimal or maximal acute inflammatory response.

Airway smooth muscle constriction induced by cholinergic agonists such as methacholine (MCh), which is typically increased in asthmatic patients, is regulated mainly by muscle muscarinic M3 receptors and negatively by vagal muscarinic M2 receptors. Here we evaluated basal (intrinsic) and allergen-induced (extrinsic) airway responses to MCh. We used two mouse lines selected to respond maximally (AIRmax) or minimally (AIRmin) to innate inflammatory stimuli. We found that in basal condition AIRmin mice responded more vigorously to MCh than AIRmax. Treatment with a specific M2 antagonist increased airway response of AIRmax but not of AIRmin mice. The expression of M2 receptors in the lung was significantly lower in AIRmin compared to AIRmax animals. AIRmax mice developed a more intense allergic inflammation than AIRmin, and both allergic mouse lines increased airway responses to MCh. However, gallamine treatment of allergic groups did not affect the responses to MCh. Our results confirm that low or dysfunctional M2 receptor activity is associated with increased airway responsiveness to MCh and that this trait was inherited during the selective breeding of AIRmin mice and was acquired by AIRmax mice during allergic lung inflammation.

Enhancement of Methacholine-Evoked Tracheal Contraction Induced by Bacterial Lipopolysaccharides Depends on Epithelium and Tumor Necrosis Factor

Inhaled bacterial lipopolysaccharides (LPSs) induce an acute tumour necrosis factor-alpha (TNF-α-) dependent inflammatory response in the murine airways mediated by Toll-like receptor 4 (TLR4) via the myeloid differentiation MyD88 adaptor protein pathway. However, the contractile response of the bronchial smooth muscle and the role of endogenous TNFα in this process have been elusive. We determined the in vivo respiratory pattern of C57BL/6 mice after intranasal LPS administration with or without the presence of increasing doses of methacholine (MCh). We found that LPS administration altered the basal and MCh-evoked respiratory pattern that peaked at 90 min and decreased thereafter in the next 48 h, reaching basal levels 7 days later. We investigated in controlled ex vivo condition the isometric contraction of isolated tracheal rings in response to MCh cholinergic stimulation. We observed that preincubation of the tracheal rings with LPS for 90 min enhanced the subsequent MCh-induced contractile response (hyperreactivity), which was prevented by prior neutralization of TNFα with a specific antibody. Furthermore, hyperreactivity induced by LPS depended on an intact epithelium, whereas hyperreactivity induced by TNFα was well maintained in the absence of epithelium. Finally, the enhanced contractile response to MCh induced by LPS when compared with control mice was not observed in tracheal rings from TLR4- or TNF- or TNF-receptor-deficient mice. We conclude that bacterial endotoxin-mediated hyperreactivity of isolated tracheal rings to MCh depends upon TLR4 integrity that signals the activation of epithelium, which release endogenous TNFα.

CpG-ODN Shapes Alum Adjuvant Activity Signaling via MyD88 and IL-10

Aluminum-containing adjuvants usually referred as Alum are considered as T helper type-2 (Th2) adjuvants, while agonists of toll-like receptors (TLRs) are viewed as adjuvants that favor Th1/Th17 immunity. Alum has been used in numerous vaccine formulations; however, its undesired pro-Th2 adjuvant activity constitutes a caveat for Alum-based vaccines. Combining Alum with TLR-dependent, pro-Th1/Th17 adjuvants might dampen the pro-Th2 activity and improve the effectiveness of vaccine formulations. Here, using the ovalbumin (OVA) model of allergic lung inflammation, we found that sensitization with the synthetic TLR9 agonist, which is composed of oligodeoxynucleotides containing CpG motifs adsorbed to Alum, inhibited the development of OVA-induced lung allergic Th2 responses without shifting toward a Th1 pattern. The conversion of T cell immunity from the polarized allergic Th2 response to a non-polarized form by sensitization with OVA/Alum/CpG was dependent on MyD88 signaling in myeloid cells. Notably, sensitization of IL-10-deficient mice with OVA/Alum/CpG resulted in the development of neutrophilic lung inflammation associated with IFNγ production. However, in IL-10/IL-12-deficient mice, it resulted in neutrophilic inflammation dominated by IL-17 production. We conclude that OVA/Alum/CpG sensitization signaling via MyD88 and IL-10 molecules results in non-polarized immunity. Conversely, OVA/Alum/CpG sensitization in presence of MyD88 but absence of IL-10 or IL-10/IL-12 molecules results, respectively, in neutrophilic inflammation associated with IFNγ or IL-17 production. Our work provides novel OVA models of lung inflammation and suggests that Alum/CpG-based formulations might be of potential use in anti-allergic or anti-infectious processes.

Exposure to Aedes aegypti Bites Induces a Mixed-Type Allergic Response following Salivary Antigens Challenge in Mice.

Classical studies have shown that Aedes aegypti salivary secretion is responsible for the sensitization to mosquito bites and many of the components present in saliva are immunogenic and capable of inducing an intense immune response. Therefore, we have characterized a murine model of adjuvant-free systemic allergy induced by natural exposure to mosquito bites. BALB/c mice were sensitized by exposure to A. aegypti mosquito bites and intranasally challenged with phosphate-buffered saline only or the mosquito’s salivary gland extract (SGE). Blood, bronchoalveolar lavage (BAL) and lung were collected and evaluated for cellularity, histopathological analyses, cytokines and antibody determination. Respiratory pattern was analyzed by Penh measurements and tracheal segments were obtained to study in vitro reactivity to methacholine. BAL recovered from sensitized mice following challenge with SGE showed an increased number of eosinophils and Th2 cytokines such as IL-4, IL-5 and IL-13. Peribronchoalveolar eosinophil infiltration, mucus and collagen were also observed in lung parenchyma of sensitized mice, suggesting the development of a typical Th2 response. However, the antibody profile in serum of these mice evidenced a mixed-type response with presence of both, IgG1/IgE (Th2-related) and IgG2a (Th1-related) isotypes. In addition, changes in breathing pattern and tracheal reactivity to methacholine were not found. Taken together, our results show that A. aegypti bites trigger an atypical allergic reaction, with some classical cellular and soluble Th2 components in the lung, but also systemic Th1 and Th2 antibody isotypes and no change in either the respiratory pattern or the trachea responsiveness to agonist.