Modulation of Allergic Sensitization and Allergic Inflammation by Staphylococcus aureus Enterotoxin B in an Ovalbumin Mouse Model

The superantigen Staphylococcus aureus (S. aureus) enterotoxin B (SEB) has been proposed a central player in the associations between S. aureus nasal colonization and the development of allergic asthma. Previously, SEB has been shown to aggravate allergic sensitization and allergic airway inflammation (AAI) in experimental mouse models. Aiming at understanding the underlying immunological mechanisms, we tested the hypothesis that intranasal (i.n.) SEB-treatment divergently modulates AAI depending on the timing and intensity of the SEB-encounter. In an ovalbumin-mediated mouse model of AAI, we treated mice i.n. with 50 ng or 500 ng SEB either together with the allergic challenge or prior to the peripheral sensitization. We observed SEB to affect different hallmark parameters of AAI depending on the timing and the dose of treatment. SEB administered i.n. together with the allergic challenge significantly modulated respiratory leukocyte accumulation, intensified lymphocyte activation and, at the higher dose, induced a strong type-1 and pro-inflammatory cytokine response and alleviated airway hyperreactivity in AAI. SEB administered i.n. prior to the allergic sensitization at the lower dose significantly boosted the specific IgE response while administration of the higher dose led to a significantly reduced recruitment of immune cells, including eosinophils, to the respiratory tract and to a significantly dampened Th-2 cytokine response without inducing a Th-1 or pro-inflammatory response. We show a remarkably versatile potential for SEB to either aggravate or alleviate different parameters of allergic sensitization and AAI. Our study thereby not only highlights the complexity of the associations between S. aureus and allergic asthma but possibly even points at prophylactic and therapeutic pathways

In vivo Neutralization of Pro-inflammatory Cytokines During Secondary Streptococcus pneumoniae Infection Post Influenza A Virus Infection

An overt pro-inflammatory immune response is a key factor contributing to lethal pneumococcal infection in an influenza pre-infected host and represents a potential target for therapeutic intervention. However, there is a paucity of knowledge about the level of contribution of individual cytokines. Based on the predictions of our previous mathematical modeling approach, the potential benefit of IFN-γ- and/or IL-6-specific antibody-mediated cytokine neutralization was explored in C57BL/6 mice infected with the influenza A/PR/8/34 strain, which were subsequently infected with the Streptococcus pneumoniae strain TIGR4 on day 7 post influenza. While single IL-6 neutralization had no effect on respiratory bacterial clearance, single IFN-γ neutralization enhanced local bacterial clearance in the lungs. Concomitant neutralization of IFN-γ and IL-6 significantly reduced the degree of pneumonia as well as bacteremia compared to the control group, indicating a positive effect for the host during secondary bacterial infection. The results of our model-driven experimental study reveal that the predicted therapeutic value of IFN-γ and IL-6 neutralization in secondary pneumococcal infection following influenza infection is tightly dependent on the experimental protocol while at the same time paving the way toward the development of effective immune therapies

Infection-Associated Mechanisms of Neuro-Inflammation and Neuro-Immune Crosstalk in Chronic Respiratory Diseases

Chronic obstructive airway diseases are characterized by airflow obstruction and airflow limitation as well as chronic airway inflammation. Especially bronchial asthma and chronic obstructive pulmonary disease (COPD) cause considerable morbidity and mortality worldwide, can be difficult to treat, and ultimately lack cures. While there are substantial knowledge gaps with respect to disease pathophysiology, our awareness of the role of neurological and neuro-immunological processes in the development of symptoms, the progression, and the outcome of these chronic obstructive respiratory diseases, is growing. Likewise, the role of pathogenic and colonizing microorganisms of the respiratory tract in the development and manifestation of asthma and COPD is increasingly appreciated. However, their role remains poorly understood with respect to the underlying mechanisms. Common bacteria and viruses causing respiratory infections and exacerbations of chronic obstructive respiratory diseases have also been implicated to affect the local neuro-immune crosstalk. In this review, we provide an overview of previously described neuro-immune interactions in asthma, COPD, and respiratory infections that support the hypothesis of a neuro-immunological component in the interplay between chronic obstructive respiratory diseases, respiratory infections, and respiratory microbial colonization

The Role of <i>Staphylococcus aureus</i> and Its Toxins in the Pathogenesis of Allergic Asthma

Bronchial asthma is one of the most common chronic diseases worldwide and affects more than 300 million patients. Allergic asthma affects the majority of asthmatic children as well as approximately 50% of adult asthmatics. It is characterized by a Th2-mediated immune response against aeroallergens. Many aspects of the overall pathophysiology are known, while the underlying mechanisms and predisposing factors remain largely elusive today. Over the last decade, respiratory colonization with Staphylococcus aureus (S. aureus), a Gram-positive facultative bacterial pathogen, came into focus as a risk factor for the development of atopic respiratory diseases. More than 30% of the world’s population is constantly colonized with S. aureus in their nasopharynx. This colonization is mostly asymptomatic, but in immunocompromised patients, it can lead to serious complications including pneumonia, sepsis, or even death. S. aureus is known for its ability to produce a wide range of proteins including toxins, serine-protease-like proteins, and protein A. In this review, we provide an overview of the current knowledge about the pathophysiology of allergic asthma and to what extent it can be affected by different toxins produced by S. aureus. Intensifying this knowledge might lead to new preventive strategies for atopic respiratory diseases

Respiratory Influenza A Virus Infection Triggers Local and Systemic Natural Killer Cell Activation via Toll-Like Receptor 7

The innate immune system senses influenza A virus (IAV) through different pathogen-recognition receptors including Toll-like receptor 7 (TLR7). Downstream of viral recognition natural killer (NK) cells are activated as part of the anti-IAV immune response. Despite the known decisive role of TLR7 for NK cell activation by therapeutic immunostimulatory RNAs, the contribution of TLR7 to the NK cell response following IAV infection has not been addressed. We have analyzed lung cytokine responses as well as the activation, interferon (IFN)-γ production, and cytotoxicity of lung and splenic NK cells following sublethal respiratory IAV infection in wild-type and TLR7ko mice. Early airway IFN-γ levels as well as the induction of lung NK cell CD69 expression and IFN-γ production in response to IAV infection were significantly attenuated in TLR7-deficient hosts. Strikingly, respiratory IAV infection also primed splenic NK cells for IFN-γ production, degranulation, and target cell lysis, all of which were fully dependent on TLR7. At the same time, lung type I IFN levels were significantly reduced in TLR7ko mice early following IAV infection, displaying a potential upstream mechanism of the attenuated NK cell activation observed. Taken together, our data clearly demonstrate a specific role for TLR7 signaling in local and systemic NK cell activation following respiratory IAV infection despite the presence of redundant innate IAV-recognition pathways

Resolved Influenza A Virus Infection Has Extended Effects on Lung Homeostasis and Attenuates Allergic Airway Inflammation in a Mouse Model.

Allergic airway inflammation (AAI) involves T helper cell type 2 (Th2) and pro-inflammatory responses to aeroallergens and many predisposing factors remain elusive. Influenza A virus (IAV) is a major human pathogen that causes acute respiratory infections and induces specific immune responses essential for viral clearance and resolution of the infection. Beyond acute infection, IAV has been shown to persistently affect lung homeostasis and respiratory immunity. Here we asked how resolved IAV infection affects subsequently induced AAI. Mice infected with a sublethal dose of IAV were sensitized and challenged in an ovalbumin mediated mouse model for AAI after resolution of the acute viral infection. Histological changes, respiratory leukocytes, cytokines and airway hyperreactivity were analyzed in resolved IAV infection alone and in AAI with and without previous IAV infection. More than five weeks after infection, we detected persistent pneumonia with increased activated CD4+ and CD8+ lymphocytes as well as dendritic cells and MHCII expressing macrophages in the lung. Resolved IAV infection significantly affected subsequently induced AAI on different levels including morphological changes, respiratory leukocytes and lymphocytes as well as the pro-inflammatory cytokine responses, which was clearly diminished. We conclude that IAV has exceptional persisting effects on respiratory immunity with substantial consequences for subsequently induced AAI

The STING activator c-di-AMP exerts superior adjuvant properties than the formulation poly(I:C)/CpG after subcutaneous vaccination with soluble protein antigen or DEC-205-mediated antigen targeting to dendritic cells.

Vaccination is the most efficient strategy to protect from infectious diseases and the induction of a protective immune response not only depends on the nature of the antigen, but is also influenced by the vaccination strategy and the co-administration of adjuvants. Therefore, the precise monitoring of adjuvant candidates and their immune modulatory properties is a crucial step in vaccine development. Here, one central aspect is the induction of appropriate humoral and cellular effector mechanisms. In our study we performed a direct comparison of two promising candidates in adjuvant development, the STING activator bis-(3,5)-cyclic dimeric adenosine monophosphate (c-di-AMP) and the Toll-like receptor ligand formulation poly(I:C)/CpG. These were evaluated in C57BL/6 mice using the model antigen ovalbumin (OVA) in subcutaneous vaccination with soluble protein as well as in a dendritic cell (DC) targeting approach (αDEC-OVA). Strikingly, c-di-AMP as compared to poly(I:C)/CpG resulted in significantly higher antigen-specific IgG antibody levels when used in immunization with soluble OVA as well as in antigen targeting to DC. In vaccination with soluble OVA, c-di-AMP induced a significantly stronger CTL, Th1 and IFNγ-producing CD8+ memory T cell response than poly(I:C)/CpG. The response was CTL and Th1 cell dominated, a profile shared by both adjuvants. In the context of targeting OVA to DC, c-di-AMP induced significantly increased Th1 and Th2 cell responses as compared to poly(I:C)/CpG. Interestingly, the Th1 response dominated the overall T cell response only when c-di-AMP was used, indicating a distinct modulatory property of c-di-AMP when the DC targeting immunization approach was exploited. Taken together, we describe superior properties of c-di-AMP as compared to poly(I:C)/CpG in subcutaneous vaccination with soluble antigen as well as antigen targeting to DC. This indicates exceptionally effective adjuvant properties for c-di-AMP and provides compelling evidence of its potential for further adjuvant development, especially also when using DC targeting approaches

Chemical Conjugation of a Purified DEC-205-Directed Antibody with Full-Length Protein for Targeting Mouse Dendritic Cells In Vitro and In Vivo.

Targeted antigen delivery to cross-presenting dendritic cells (DC) in vivo efficiently induces T effector cell responses and displays a valuable approach in vaccine design. Antigen is delivered to DC via antibodies specific for endocytosis receptors such as DEC-205 that induce uptake, processing, and MHC class I- and II-presentation. Efficient and reliable conjugation of the desired antigen to a suitable antibody is a critical step in DC targeting and among other factors depends on the format of the antigen. Chemical conjugation of full-length protein to purified antibodies is one possible strategy. In the past, we have successfully established cross-linking of the model antigen ovalbumin (OVA) and a DEC-205-specific IgG2a antibody (αDEC-205) for in vivo DC targeting studies in mice. The first step of the protocol is the purification of the antibody from the supernatant of the NLDC (non-lymphoid dendritic cells)-145 hybridoma by affinity chromatography. The purified antibody is activated for chemical conjugation by sulfo-SMCC (sulfosuccinimidyl 4-[N-maleimidomethyl] cyclohexane-1-carboxylate) while at the same time the sulfhydryl-groups of the OVA protein are exposed through incubation with TCEP-HCl (tris (2-carboxyethyl) phosphine hydrochloride). Excess TCEP-HCl and sulfo-SMCC are removed and the antigen is mixed with the activated antibody for overnight coupling. The resulting αDEC-205/OVA conjugate is concentrated and freed from unbound OVA. Successful conjugation of OVA to αDEC-205 is verified by western blot analysis and enzyme-linked immunosorbent assay (ELISA). We have successfully used chemically crosslinked αDEC-205/OVA to induce cytotoxic T cell responses in the liver and to compare different adjuvants for their potential in inducing humoral and cellular immunity following in vivo targeting of DEC-205+ DC. Beyond that, such chemically coupled antibody/antigen conjugates offer valuable tools for the efficient induction of vaccine responses to tumor antigens and have been proven to be superior to classical immunization approaches regarding the prevention and therapy of various types of tumors