Thomas Naessens

Polyelectrolyte Microcapsules as Antigen Delivery Vehicles To Dendritic Cells: Uptake, Processing, and Cross‐Presentation of Encapsulated Antigens

Degradable polyelectrolyte microcapsules (PMs; see picture) as antigen delivery vehicles are taken up by dendritic cells (DCs) by macropinocytosis. Following uptake, the shell of the microcapsules ruptures, resulting in the invasion of the capsules by the cellular cytoplasm, thus allowing DCs to efficiently process encapsulated antigen.

Type I IFN Counteracts the Induction of Antigen-Specific Immune Responses by Lipid-Based Delivery of mRNA Vaccines

The use of DNA and viral vector-based vaccines for the induction of cellular immune responses is increasingly gaining interest. However, concerns have been raised regarding the safety of these immunization strategies. Due to the lack of their genome integration, mRNA-based vaccines have emerged as a promising alternative. In this study, we evaluated the potency of antigen-encoding mRNA complexed with the cationic lipid 1,2-dioleoyl-3trimethylammonium-propane/1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOTAP/DOPE ) as a novel vaccination approach. We demonstrate that subcutaneous immunization of mice with mRNA encoding the HIV-1 antigen Gag complexed with DOTAP/DOPE elicits antigen-specific, functional T cell responses resulting in specific killing of Gag peptide-pulsed cells and the induction of humoral responses. In addition, we show that DOTAP/DOPE complexed antigen-encoding mRNA displays immune-activating properties characterized by secretion of type I interferon (IFN) and the recruitment of proinflammatory monocytes to the draining lymph nodes. Finally, we demonstrate that type I IFN inhibit the expression of DOTAP/DOPE complexed antigen-encoding mRNA and the subsequent induction of antigen-specific immune responses. These results are of high relevance as they will stimulate the design and development of improved mRNA-based vaccination approaches.

Biodegradable Polyelectrolyte Microcapsules: Antigen Delivery Tools with Th17 Skewing Activity after Pulmonary Delivery

Because of their large surface area, the lungs appear an attractive route for noninvasive vaccine delivery, harboring the potential to induce local mucosal immune responses in addition to systemic immunity. To evoke adaptive immunity, Ags require the addition of adjuvants that not only enhance the strength of the immune response but also determine the type of response elicited. In this study, we evaluate the adjuvant characteristics of polyelectrolyte microcapsules (PEMs) consisting of the biopolymers dextran-sulfate and poly-l-arginine. PEMs form an entirely new class of microcapsules that are generated by the sequential adsorption of oppositely charged polymers (polyelectrolytes) onto a sacrificial colloidal template, which is subsequently dissolved leaving a hollow microcapsule surrounded by a thin shell. Following intratracheal instillation, PEMs were not only efficiently taken up by APCs but also enhanced their activation status. Pulmonary adaptive immune responses were characterized by the induction of a strongly Th17-polarized response. When compared with a mixture of soluble Ag with empty microcapsules, Ag encapsulation significantly enhanced the strength of this local mucosal response. Given their unique property to selectively generate Th17-polarized immune responses, PEMs may become of significant interest in the development of effective vaccines against fungal and bacterial species.

Molecular structure of the Mycobacterium tuberculosis virulence factor, mycolic acid, determines the elicited inflammatory pattern

Mycolic acids (MAs) occur in the cell wall of Mycobacterium tuberculosis as variable mixtures of different classes and chain lengths. Here, we address the relationship between the structure and its inflammatory function of this virulence factor using single synthetic MA isomers, differing in oxygenation class and cis‐ versus α‐methyl‐trans proximal cyclopropane orientation. Analysis of bronchoalveolar inflammation, lung histopathology and alveolar macrophage transcription revealed a strong dependence on these meromycolic chemistries of mouse pulmonary inflammation in response to intratracheal treatments with MAs. Whereas α‐MA was inert, oxygenated methoxy‐ and keto‐MA with cis‐cyclopropane stereochemistry elicited solid to mild inflammatory responses respectively. In trans‐cyclopropane orientation, methoxy‐MA partially lost its inflammatory activity and keto‐MA exerted anti‐inflammatory alternative activation of alveolar macrophages and counteracted cis‐methoxy‐MA induced airway inflammation. The differential innate immune activities of MAs demonstrated here, dependent on oxygenation class and cis versus α‐methyl‐trans cyclopropane chemistry, identify a novel means for M. tuberculosis to steer host immune responses during infection.

Where asthma and hypersensitivity pneumonitis meet and differ: noneosinophilic severe asthma

Asthma is a type-I allergic airway disease characterized by Th(2) cells and IgE. Episodes of bronchial inflammation, eosinophilic in nature and promoting bronchoconstriction, may become chronic and lead to persistent respiratory symptoms and irreversible structural airway changes. Representative mostly of mild to moderate asthma, this clinical definition fails to account for the atypical and often more severe phenotype found in a considerable proportion of asthmatics who have increased neutrophil cell counts in the airways as a distinguishing trait. Neutrophilic inflammation is a hallmark of another type of allergic airway pathology, hypersensitivity pneumonitis. Considered as an immune counterpart of asthma, hypersensitivity pneumonitis is a prototypical type-III allergic inflammatory reaction involving the alveoli and lung interstitium, steered by Th(1) cells and IgG and, in its chronic form, accompanied by fibrosis. Although pathologically very different and commonly approached as separate disorders, as discussed in this review, clinical studies as well as data from animal models reveal undeniable parallels between both airway diseases. Danger signaling elicited by the allergenic agent or by accompanying microbial patterns emerges as critical in enabling immune sensitization and in determining the type of sensitization and ensuing allergic disease. On this basis, we propose that asthma allergens cause severe noneosinophilic asthma because of sensitization in the presence of hypersensitivity pneumonitis-promoting danger signaling.

Inflammatory signatures for eosinophilic vs. neutrophilic allergic pulmonary inflammation reveal critical regulatory checkpoints

Contrary to the T-helper (Th)-2 bias and eosinophil-dominated bronchial inflammation encountered in most asthmatic subjects, other patients may exhibit neutrophil-predominant asthma subphenotypes, along with Th-1 and Th-17 cells. However, the etiology of many neutrophil-dominated asthma subphenotypes remains ill-understood, in part due to a lack of appropriate experimental models. To better understand the distinct immune-pathological features of eosinophilic vs. neutrophilic asthma types, we developed an ovalbumin (OVA)-based mouse model of neutrophil-dominated allergic pulmonary inflammation. Consequently, we probed for particular inflammatory signatures and checkpoints underlying the immune pathology in this new model, as well as in a conventional, eosinophil-dominated asthma model. Briefly, mice were OVA sensitized using either aluminum hydroxide (alum) or complete Freunds adjuvants, followed by OVA aerosol challenge. T-cell, granulocyte, and inflammatory mediator profiles were determined, along with alveolar macrophage genomewide transcriptome profiling. In contrast to the Th-2-dominated phenotype provoked by alum, OVA/ complete Freunds adjuvants adjuvant-based sensitization, followed by allergen challenge, elicited a pulmonary inflammation that was poorly controlled by dexamethasone, and in which Th-1 and Th-17 cells additionally participated. Analysis of the overall pulmonary and alveolar macrophage inflammatory mediator profiles revealed remarkable similarities between both models. Nevertheless, we observed pronounced differences in the IL-12/IFN-γ axis and its control by IL-18 and IL-18 binding protein, but also in macrophage arachidonic acid metabolism and expression of T-cell instructive ligands. These differential signatures, superimposed onto a generic inflammatory signature, denote distinctive inflammatory checkpoints potentially involved in orchestrating neutrophil-dominated asthma.

Neutralizing TNFα restores glucocorticoid sensitivity in a mouse model of neutrophilic airway inflammation

Asthma is a heterogeneous disorder, evidenced by distinct types of inflammation resulting in different responsiveness to therapy with glucocorticoids (GCs). Tumor necrosis factor α (TNFα) is involved in asthma pathogenesis, but anti-TNFα therapies have not proven broadly effective. The effects of anti-TNFα treatment on steroid resistance have never been assessed. We investigated the role of TNFα blockade using etanercept in the responsiveness to GCs in two ovalbumin-based mouse models of airway hyperinflammation. The first model is GC sensitive and T helper type 2 (Th2)/eosinophil driven, whereas the second reflects GC-insensitive, Th1/neutrophil-predominant asthma subphenotypes. We found that TNFα blockade restores the therapeutic effects of GCs in the GC-insensitive model. An adoptive transfer indicated that the TNFα-induced GC insensitivity occurs in the non-myeloid compartment. Early during airway hyperinflammation, mice are GC insensitive specifically at the level of thymic stromal lymphopoietin (Tslp) transcriptional repression, and this insensitivity is reverted when TNFα is neutralized. Interestingly, TSLP knockout mice displayed increased inflammation in the GC-insensitive model, suggesting a limited therapeutic application of TSLP-neutralizing antibodies in subsets of patients suffering from Th2-mediated asthma. In conclusion, we demonstrate that TNFα reduces the responsiveness to GCs in a mouse model of neutrophilic airway inflammation. Thus antagonizing TNFα may offer a new strategy for therapeutic intervention in GC-resistant asthma.

Innate Imprinting of Murine Resident Alveolar Macrophages by Allergic Bronchial Inflammation Causes a Switch from Hypoinflammatory to Hyperinflammatory Reactivity

Resident alveolar macrophages (rAMs) residing in the bronchoalveolar lumen of the airways play an important role in limiting excessive inflammatory responses in the respiratory tract. High phagocytic activity along with hyporesponsiveness to inflammatory insults and lack of autonomous IFN-β production are crucial assets in this regulatory function. Using a mouse model of asthma, we analyzed the fate of rAMs both during and after allergic bronchial inflammation. Although nearly indistinguishable phenotypically from naïve rAMs, postinflammation rAMs exhibited a strongly reduced basal phagocytic capacity, accompanied by a markedly increased inflammatory reactivity to Toll-like receptors TLR-3 (poly I:C), TLR-4 [lipopolysaccharide (LPS)], and TLR-7 (imiquimod). Importantly, after inflammation, rAMs exhibited a switch from an IFN-β-defective to an IFN-β-competent phenotype, thus indicating the occurrence of a new, inflammatory-released rAM population in the postallergic lung. Analysis of rAM turnover revealed a rapid disappearance of naïve rAMs after the onset of inflammation. This inflammation-induced rAM turnover is critical for the development of the hyperinflammatory rAM phenotype observed after clearance of bronchial inflammation. These data document a novel mechanism of innate imprinting in which noninfectious bronchial inflammation causes alveolar macrophages to acquire a highly modified innate reactivity. The resulting increase in secretion of inflammatory mediators on TLR stimulation implies a role for this phenomenon of innate imprinting in the increased sensitivity of postallergic lungs to inflammatory insults.

Hybrid pulmonary surfactant-coated nanogels mediate efficient in vivo delivery of siRNA to murine alveolar macrophages

The local delivery of small interfering RNA (siRNA) to the lungs may provide a therapeutic solution to a range of pulmonary disorders. Resident alveolar macrophages (rAM) in the bronchoalveolar lumen play a critical role in lung inflammatory responses and therefore constitute a particularly attractive target for siRNA therapeutics. However, achieving efficient gene silencing in the lung while avoiding pulmonary toxicity requires appropriate formulation of siRNA in functional nanocarriers. In this study, we evaluated pulmonary surfactant-coated dextran nanogels for the delivery of siRNA to rAM upon pharyngeal aspiration in BALB/c mice. Both the surfactant-coated and uncoated nanogels achieved high levels of siRNA uptake in rAM, yet only the surfactant-coated formulation could significantly reduce gene expression on the protein level. Surfactant-coated nanogels induced a profound downregulation of target mRNA levels, reaching 70% knockdown with ~1mgkg(-1) siRNA dose. In addition, only mild acute pro-inflammatory cytokine and chemokine responses were detected one day after nanoparticle aspiration, accompanied by a moderate neutrophil infiltration in the bronchoalveolar lumen. The latter could be substantially reduced by removal of excess surfactant from the formulation. Overall, our hybrid core-shell nanoparticles have demonstrated safe and effective siRNA delivery to rAM, providing a new therapeutic approach for treatment of inflammatory pathologies in the lung.

E-cadherin expression in macrophages dampens their inflammatory responsiveness in vitro, but does not modulate M2-regulated pathologies in vivo

IL-4/IL-13-induced alternatively activated macrophages (M(IL-4/IL-13), AAMs or M2) are known to express E-cadherin, enabling them to engage in heterotypic cellular interactions and IL-4-driven macrophage fusion in vitro. Here we show that E-cadherin overexpression in Raw 264.7 macrophages inhibits their inflammatory response to LPS stimulation, as demonstrated by a reduced secretion of inflammatory mediators like interleukin (IL)-6, tumor necrosis factor (TNF) and nitric oxide (NO). To study the function of E-cadherin in M(IL-4/IL-13) macrophages in vivo, we generated macrophage-specific E-cadherin-deficient C57BL/6 mice. Using this new tool, we analyzed immunological parameters during two typical AAM-associated Th2-driven diseases and assessed Th2-associated granuloma formation. Although E-cadherin is strongly induced in AAMs during Taenia crassiceps helminth infections and allergic airway inflammation, its deletion in macrophages does not affect the course of both Th2 cytokine-driven diseases. Moreover, macrophage E-cadherin expression is largely redundant for granuloma formation around Schistosoma mansoni ova. Overall, we conclude that E-cadherin is a valuable AAM marker which suppresses the inflammatory response when overexpressed. Yet E-cadherin deletion in macrophages does not affect M(LPS+IFNγ) and M(IL-4) polarization in vitro, nor in vivo macrophage function, at least in the conditions tested.