Moyar Q. Ge

Adeno-Associated Virus 9-Mediated Airway Expression of Antibody Protects Old and Immunodeficient Mice against Influenza Virus

ABSTRACT Influenza causes serious and sometimes fatal disease in individuals at risk due to advanced age or immunodeficiencies. Despite progress in the development of seasonal influenza vaccines, vaccine efficacy in elderly and immunocompromised individuals remains low. We recently developed a passive immunization strategy using an adeno-associated virus (AAV) vector to deliver a neutralizing anti-influenza antibody at the site of infection, the nasal airways. Here we show that young, old, and immunodeficient (severe combined immunodeficient [SCID]) mice that were treated intranasally with AAV9 vector expressing a modified version of the broadly neutralizing anti-influenza antibody FI6 were protected and exhibited no signs of disease following an intranasal challenge with the mouse-adapted H1N1 influenza strain A/Puerto Rico/8/1934(H1N1) (PR8) (Mt. Sinai strain). Nonvaccinated mice succumbed to the PR8 challenge due to severe weight loss. We propose that airway-directed AAV9 passive immunization against airborne infectious agents may be beneficial in elderly and immunocompromised patients, for whom there still exists an unmet need for effective vaccination against influenza.

Deficiency of Melanoma Differentiation–associated Protein 5 Results in Exacerbated Chronic Postviral Lung Inflammation

RATIONALE Respiratory viral infections can result in the establishment of chronic lung diseases. Understanding the early innate immune mechanisms that participate in the development of chronic postviral lung disease may reveal new targets for therapeutic intervention. The intracellular viral sensor protein melanoma differentiation-associated protein 5 (MDA5) sustains the acute immune response to Sendai virus, a mouse pathogen that causes chronic lung inflammation, but its role in the development of postviral chronic lung disease is unknown. OBJECTIVES To establish the role of MDA5 in the development of chronic lung disease. METHODS MDA5-deficient or control mice were infected with Sendai virus. The acute inflammatory response was evaluated by profiling chemokine and cytokine expression and by characterizing the composition of the cellular infiltrate. The impact of MDA5 on chronic lung pathology and function was evaluated through histological studies, degree of oxygen saturation, and responsiveness to carbachol. MEASUREMENTS AND MAIN RESULTS MDA5 deficiency resulted in normal virus replication and in a distinct profile of chemokines and cytokines that associated with acute lung neutropenia and enhanced accumulation of alternatively activated macrophages. Diminished expression of neutrophil-recruiting chemokines was also observed in cells infected with influenza virus, suggesting a key role of MDA5 in driving the early accumulation of neutrophils at the infection site. The biased acute inflammatory response of MDA5-deficient mice led to an enhanced chronic lung inflammation, epithelial cell hyperplasia, airway hyperreactivity, and diminished blood oxygen saturation. CONCLUSIONS MDA5 modulates the development of chronic lung inflammation by regulating the early inflammatory response in the lung.

Cutting edge: Role of NK cells and surfactant protein D in dendritic cell lymph node homing: Effects of ozone exposure

The roles of NK cells, surfactant protein D (SP-D), and IFN-γ, as well as the effect of ozone (O3) inhalation, were studied on recirculation of pulmonary dendritic cells (DC) to the mediastinal lymph nodes. O3 exposure and lack of SP-D reduced NK cell IFN-γ and lung tissue CCL21 mRNA expression and impaired DC homing to the mediastinal lymph nodes. Notably, addition of recombinant SP-D to naive mononuclear cells stimulated IFN-γ release in vitro. Because NKp46, a glycosylated membrane receptor, was necessary for dose-dependent SP-D binding to NK cells in vitro and DC migration in vivo, we speculate that SP-D may constitutively stimulate IFN-γ production by NK cells, possibly via NKp46. This mechanism could then initiate the IFN-γ/IL-12 feedback circuit, a key amplifier of DC lymph node homing. Inhibition of this process during an acute inflammatory response causes DC retention in the peripheral lung tissue and contributes to injury.

mTORC2 regulates multiple aspects of NKT-cell development and function

Invariant NKT (iNKT) cells bridge innate and adaptive immunity by rapidly secreting cytokines and lysing targets following TCR recognition of lipid antigens. Based on their ability to secrete IFN‐γ, IL‐4 and IL‐17A, iNKT‐cells are classified as NKT‐1, NKT‐2, and NKT‐17 subsets, respectively. The molecular pathways regulating iNKT‐cell fate are not fully defined. Recent studies implicate Rictor, a required component of mTORC2, in the development of select iNKT‐cell subsets, however these reports are conflicting. To resolve these questions, we used Rictorfl/fl CD4cre+ mice and found that Rictor is required for NKT‐17 cell development and normal iNKT‐cell cytolytic function. Conversely, Rictor is not absolutely required for IL‐4 and IFN‐γ production as peripheral iNKT‐cells make copious amounts of these cytokines. Overall iNKT‐cell numbers are dramatically reduced in the absence of Rictor. We provide data indicating Rictor regulates cell survival as well as proliferation of developing and mature iNKT‐cells. Thus, mTORC2 regulates multiple aspects of iNKT‐cell development and function.

Patching it together: Epicutaneous vaccination with heat labile Escherichia coli toxin against birch pollen allergy

In immunotherapy, a person’s immune system is utilized to induce or suppress specific immune responses that are needed for the prevention or treatment of disease. This requires the use of vaccines (biological preparations of disease-causing agents) that elicit a coordinated response by the adaptive and innate immune systems, with professional antigen-presenting cells such as dendritic cells playing a crucial role. Successful immunotherapy results in the induction of broad and specific immune responses involving B and/or T cells depending on vaccine design (1). Vaccine development is considered among the greatest achievements in the history of medicine (2); they can be applied in the treatment of allergic and autoimmune diseases, organ transplantation, chronic infection, and even cancer (3, 4). Allergen-specific immunotherapy (AIT) entails administration of increasing doses of allergens in an attempt to restore tolerance and reduce allergic symptoms, once a patient has identified the root cause(s) of their allergy. The allergen can be given intramuscularly, subcutaneously, sublingually, orally, or intralymphatically. The doses of allergen are increased as the patient’s tolerance grows over time (5). AIT has been used for IgE-mediated allergic diseases since the early 1900s (3, 6–11), although it remains fraught with problems (3, 6–11). For example, it is difficult to produce a strictly regulated composition for the allergenic compounds and adjuvants used, or to establish consensus in regard to the doses, intervals, and length of application. The danger of anaphylaxis occurring during administration of AIT also remains high. When successful however, AIT treats and prevents development of allergic asthma, rhinitis, and venom-induced anaphylaxis and was demonstrated to prevent sensitization with new allergens for up to 12 years (3, 12). Therefore, the quest to develop safer and more effective AIT continues today. Induction of allergen tolerance requires a decrease in IgE and increase in allergen-specific IgG levels (13). In this issue of Allergy, Cabauatan et al. describe birch pollen (rBet v 1)specific AIT in a guinea pig model, using a patch delivery system (originally developed for epicutaneous vaccination against traveler’s diarrhea). They chose guinea pigs to work with because their skin has similar features to human skin and the patch delivery system of the same size is used for humans (14). To enhance IgG levels, Cabauatan and colleagues applied heat-labile Escherichia coli (E. coli) toxin (LT) as adjuvant together with the rBet v 1 birch allergen in the skin patch and compared the effects with a traditional alum-adsorbed rBet v 1 vaccine given subcutaneously. Patch vaccination with a high allergen dose induced relevant allergen-specific IgG responses (similar to that of the subcutaneous immunization) but only when the patch contained LT. These responses were detected on day 43 after only three vaccinations, even though outbred guinea pigs might be poor responders for the rBet v 1 allergen. During successful allergen-specific immunotherapy, changes in IgG and in particular in IgG4 can result in a 10to 100-fold reduction in the allergen-specific IgE/IgG4 ratio (15). IgG4 is a nonproinflammatory immunoglobulin that is uniquely capable to form bispecific and functionally monovalent antibodies through Fab arm exchange (16), has low affinity for activating Fcc receptors, and cannot activate complement. Allergen-specific IgG4 protects against allergic responses by competitively sequestering the allergen from IgE binding (13, 17, 18). It is significant therefore that the LT adjuvant in this paper was essential for the induction of allergen-specific IgG. According to the authors, in an unpublished clinical study epicutaneous application of a high dose of rBet v 1 (without adjuvant) only induced T-cell responses but no relevant allergen-specific IgG. The authors here demonstrate that sera derived from the guinea pigs with robust rBet v 1specific IgG responses inhibited the binding of allergic patients’ IgE to the rBet v 1 allergen. This elegant result indicated that patch vaccination using LT as adjuvant can induce allergen-specific blocking IgG. By changing the ratio of allergen-specific IgE and IgG, effective AIT should desensitize the high-affinity IgE-receptor (FceRI)-bearing mast cells and basophils, decrease the numbers and activity of eosinophils and basophils in the circulation and mast cells in mucosal allergic tissues, and induce regulatory Tand B-cell activation (reviewed in (4)). To assess the clinical significance of the findings of Cabauatan and colleagues, it would be important to verify whether allergen-specific patch vaccination with LT would induce these changes. The heat-labile enterotoxigenic E. coli enterotoxin (LT) contains an ‘A’ and a pentameric ‘B’ subunit (AB5). The highly enterotoxic A subunit has ADP-ribosyltransferase activity. It activates the Gsa component of adenylate cyclase leading to elevation of intracellular cAMP, activation of cAMP-dependent PKA, and inhibition of NF-jB-dependent transcription of proinflammatory cytokines (such as TNF-a). The A subunit is delivered into the cell by the nontoxic B pentamer subunit. Given the toxic nature of the A subunit, for vaccination purposes only the engineered B subunit and various mutant derivatives are used. The main receptors for LT are the ubiquitous gangliosides, but it also binds TLR-2

Blomia tropicalis –Specific TCR Transgenic Th2 Cells Induce Inducible BALT and Severe Asthma in Mice by an IL-4/IL-13–Dependent Mechanism

Previous studies have highlighted the importance of lung-draining lymph nodes in the respiratory allergic immune response, whereas the lung parenchymal immune system has been largely neglected. We describe a new in vivo model of respiratory sensitization to Blomia tropicalis, the principal asthma allergen in the tropics, in which the immune response is focused on the lung parenchyma by transfer of Th2 cells from a novel TCR transgenic mouse, specific for the major B. tropicalis allergen Blo t 5, that targets the lung rather than the draining lymph nodes. Transfer of highly polarized transgenic CD4 effector Th2 cells, termed BT-II, followed by repeated inhalation of Blo t 5 expands these cells in the lung >100-fold, and subsequent Blo t 5 challenge induced decreased body temperature, reduction in movement, and a fall in specific lung compliance unseen in conventional mouse asthma models following a physiological allergen challenge. These mice exhibit lung eosinophilia; smooth muscle cell, collagen, and goblet cell hyperplasia; hyper IgE syndrome; mucus plugging; and extensive inducible BALT. In addition, there is a fall in total lung volume and forced expiratory volume at 100 ms. These pathophysiological changes were substantially reduced and, in some cases, completely abolished by administration of neutralizing mAbs specific for IL-4 and IL-13 on weeks 1, 2, and 3. This IL-4/IL-13–dependent inducible BALT model will be useful for investigating the pathophysiological mechanisms that underlie asthma and the development of more effective drugs for treating severe asthma.

Oxidative damage of SP-D abolishes control of eosinophil extracellular DNA trap formation

The asthmatic airways are highly susceptible to inflammatory injury by air pollutants such as ozone (O3), characterized by enhanced activation of eosinophilic granulocytes and a failure of immune protective mechanisms. Eosinophil activation during asthma exacerbation contributes to the proinflammatory oxidative stress by high levels of nitric oxide (NO) production and extracellular DNA release. Surfactant protein‐D (SP‐D), an epithelial cell product of the airways, is a critical immune regulatory molecule with a multimeric structure susceptible to oxidative modifications. Using recombinant proteins and confocal imaging, we demonstrate here that SP‐D directly bound to the membrane and inhibited extracellular DNA trap formation by human and murine eosinophils in a concentration and carbohydrate‐dependent manner. Combined allergic airway sensitization and O3 exposure heightened eosinophilia and nos2 mRNA (iNOS) activation in the lung tissue and S‐nitrosylation related de‐oligomerisation of SP‐D in the airways. In vitro reproduction of the iNOS action led to similar effects on SP‐D. Importantly, S‐nitrosylation abolished the ability of SP‐D to block extracellular DNA trap formation. Thus, the homeostatic negative regulatory feedback between SP‐D and eosinophils is destroyed by the NO‐rich oxidative lung tissue environment in asthma exacerbations.

Erratum: Cutting edge: Role of NK cells and surfactant protein D in dendritic cell lymph node homing: Effects of ozone exposure (Journal of Immunology (2016) 196 (553-557))

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