NLRP3 promotes allergic responses to birch pollen extract in a model of intranasal sensitization.

Introduction & Objective: Allergic sensitization is an essential step in the development of allergic airway inflammation to birch pollen (BP); however, this process remains to be fully elucidated. Recent scientific advances have highlighted the importance of the allergen context. In this regard, microbial patterns (PAMPs) present on BP have attracted increasing interest. As these PAMPs are recognized by specialized pattern recognition receptors (PRRs), this study aims at investigating the roles of intracellular PRRs and the inflammasome regulator NLRP3. Methods: We established a physiologically relevant intranasal and adjuvant-free sensitization procedure to study BP-induced systemic and local lung inflammation. Results: Strikingly, BP-sensitized Nlrp3-deficient mice showed significantly lower IgE levels, Th2-associated cytokines, cell infiltration into the lung, mucin production and epithelial thickening than their wild-type counterparts, which appears to be independent of inflammasome formation. Intriguingly, bone-marrow chimera revealed that expression of NLRP3 in the hematopoietic system is required to trigger an allergic response. Conclusion: Overall, this study identifies NLRP3 as an important driver of BP-induced allergic immune responses.

Mechanistic insights into silica nanoparticle-allergen interactions on antigen presenting cell function in the context of allergic reactions.

The incorporation of nanomaterials into consumer products has substantially increased in recent years, raising concerns about their safety. The inherent physicochemical properties of nanoparticles allow them to cross epithelial barriers and gain access to immunocompetent cells. Nanoparticles in cosmetic products can potentially interact with environmental allergens, forming a protein corona, and together penetrate through damaged skin. Allergen-nanoparticle interactions may influence the immune response, eventually resulting in an adverse or beneficial outcome in terms of allergic reactivity. This study determines the impact of silica nanoparticle-allergen interactions on allergic sensitization by studying the major molecular mechanisms affecting allergic responses. The major birch pollen allergen Bet v 1 was chosen as a model allergen and the birch pollen extract as a comparator. Key events in immunotoxicity including allergen uptake, processing, presentation, expression of costimulatory molecules and cytokine release were studied in human monocyte-derived dendritic cells. Using an in vivo sensitization model, murine Bet v 1-specific IgG and IgE levels were monitored. Upon the interaction of allergens with silica nanoparticles, we observed an enhanced uptake of the allergen by macropinocytosis, improved proteolytic processing, and presentation concomitant with a propensity to increase allergen-specific IgG2a and decrease IgE antibody levels. Together, these events suggest that upon nanoparticle interactions the immune response is biased towards a type 1 inflammatory profile, characterized by the upregulation of T helper 1 (Th1) cells. In conclusion, the interaction of the birch pollen allergen with silica nanoparticles will not worsen allergic sensitization, a state of type 2-inflammation, but rather seems to decrease it by skewing towards a Th1-dominated immune response.

Helicobacter pylori induces a novel form of innate immune memory via accumulation of NF-кB proteins.

Helicobacter pylori is a widespread Gram-negative pathogen involved in a variety of gastrointestinal diseases, including gastritis, ulceration, mucosa-associated lymphoid tissue (MALT) lymphoma and gastric cancer. Immune responses aimed at eradication of H. pylori often prove futile, and paradoxically play a crucial role in the degeneration of epithelial integrity and disease progression. We have previously shown that H. pylori infection of primary human monocytes increases their potential to respond to subsequent bacterial stimuli - a process that may be involved in the generation of exaggerated, yet ineffective immune responses directed against the pathogen. In this study, we show that H. pylori-induced monocyte priming is not a common feature of Gram-negative bacteria, as Acinetobacter lwoffii induces tolerance to subsequent Escherichia coli lipopolysaccharide (LPS) challenge. Although the increased reactivity of H. pylori-infected monocytes seems to be specific to H. pylori, it appears to be independent of its virulence factors Cag pathogenicity island (CagPAI), cytotoxin associated gene A (CagA), vacuolating toxin A (VacA) and γ-glutamyl transferase (γ-GT). Utilizing whole-cell proteomics complemented with biochemical signaling studies, we show that H. pylori infection of monocytes induces a unique proteomic signature compared to other pro-inflammatory priming stimuli, namely LPS and the pathobiont A. lwoffii. Contrary to these tolerance-inducing stimuli, H. pylori priming leads to accumulation of NF-кB proteins, including p65/RelA, and thus to the acquisition of a monocyte phenotype more responsive to subsequent LPS challenge. The plasticity of pro-inflammatory responses based on abundance and availability of intracellular signaling molecules may be a heretofore underappreciated form of regulating innate immune memory as well as a novel facet of the pathobiology induced by H. pylori.

The Class IIA Histone Deacetylase (HDAC) Inhibitor TMP269 Downregulates Ribosomal Proteins and Has Anti-Proliferative and Pro-Apoptotic Effects on AML Cells.

Acute myeloid leukemia (AML) is a hematopoietic malignancy characterized by altered myeloid progenitor cell proliferation and differentiation. As in many other cancers, epigenetic transcriptional repressors such as histone deacetylases (HDACs) are dysregulated in AML. Here, we investigated (1) HDAC gene expression in AML patients and in different AML cell lines and (2) the effect of treating AML cells with the specific class IIA HDAC inhibitor TMP269, by applying proteomic and comparative bioinformatic analyses. We also analyzed cell proliferation, apoptosis, and the cell-killing capacities of TMP269 in combination with venetoclax compared to azacitidine plus venetoclax, by flow cytometry. Our results demonstrate significantly overexpressed class I and class II HDAC genes in AML patients, a phenotype which is conserved in AML cell lines. In AML MOLM-13 cells, TMP269 treatment downregulated a set of ribosomal proteins which are overexpressed in AML patients at the transcriptional level. TMP269 showed anti-proliferative effects and induced additive apoptotic effects in combination with venetoclax. We conclude that TMP269 exerts anti-leukemic activity when combined with venetoclax and has potential as a therapeutic drug in AML.

Surface Functionalization of Silica Nanoparticles: Strategies to Optimize the Immune-Activating Profile of Carrier Platforms.

Silica nanoparticles (SiNPs) are generally regarded as safe and may represent an attractive carrier platform for nanomedical applications when loaded with biopharmaceuticals. Surface functionalization by different chemistries may help to optimize protein loading and may further impact uptake into the targeted tissues or cells, however, it may also alter the immunologic profile of the carrier system. In order to circumvent side effects, novel carrier candidates need to be tested thoroughly, early in their development stage within the pharmaceutical innovation pipeline, for their potential to activate or modify the immune response. Previous studies have identified surface functionalization by different chemistries as providing a plethora of modifications for optimizing efficacy of biopharmaceutical (nano)carrier platforms while maintaining an acceptable safety profile. In this study, we synthesized SiNPs and chemically functionalized them to obtain different surface characteristics to allow their application as a carrier system for allergen-specific immunotherapy. In the present study, crude natural allergen extracts are used in combination with alum instead of well-defined active pharmaceutical ingredients (APIs), such as recombinant allergen, loaded onto (nano)carrier systems with immunologically inert and stable properties in suspension. This study was motivated by the hypothesis that comparing different charge states could allow tailoring of the binding capacity of the particulate carrier system, and hence the optimization of biopharmaceutical uptake while maintaining an acceptable safety profile, which was investigated by determining the maturation of human antigen-presenting cells (APCs). The functionalized nanoparticles were characterized for primary and hydrodynamic size, polydispersity index, zeta potential, endotoxin contamination. As potential candidates for allergen-specific immunotherapy, the differently functionalized SiNPs were non-covalently coupled with a highly purified, endotoxin-free recombinant preparation of the major birch pollen allergen Bet v 1 that functioned for further immunological testing. Binding efficiencies of allergen to SiNPs was controlled to determine uptake of API. For efficacy and safety assessment, we employed human monocyte-derived dendritic cells as model for APCs to detect possible differences in the particles' APC maturation potential. Functionalization of SiNP did not affect the viability of APCs, however, the amount of API physisorbed onto the nanocarrier system, which induced enhanced uptake, mainly by macropinocytosis. We found slight differences in the maturation state of APCs for the differently functionalized SiNP-API conjugates qualifying surface functionalization as an effective instrument for optimizing the immune response towards SiNPs. This study further suggests that surface-functionalized SiNPs could be a suitable, immunologically inert vehicle for the efficient delivery of biopharmaceutical products, as evidenced here for allergen-specific immunotherapy.

Helicobacter pylori Infection of Primary Human Monocytes Boosts Subsequent Immune Responses to LPS.

Infection with Helicobacter pylori (H. pylori) affects almost half of the world's population and is a major cause of stomach cancer. Although immune cells react strongly to this gastric bacterium, H. pylori is still one of the rare pathogens that can evade elimination by the host and cause chronic inflammation. In the present study, we characterized the inflammatory response of primary human monocytes to repeated H. pylori infection and their responsiveness to an ensuing bacterial stimulus. We show that, although repeated stimulations with H. pylori do not result in an enhanced response, H. pylori-primed monocytes are hyper-responsive to an Escherichia coli-lipopolysaccharide (LPS) stimulation that takes place shortly after infection. This hyper-responsiveness to bacterial stimuli is observed upon infection with viable H. pylori only, while heat-killed H. pylori fails to boost both cytokine secretion and STAT activation in response to LPS. When the secondary challenge occurs several days after the primary infection with live bacteria, H. pylori-infected monocytes lose their hyper-responsiveness. The observation that H. pylori makes primary human monocytes more susceptible to subsequent/overlapping stimuli provides an important basis to better understand how H. pylori can maintain chronic inflammation and thus contribute to gastric cancer progression.