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Background: From early life, respiratory viruses are implicated in the development, exacerbation and persistence of respiratory conditions such as asthma. Complex dynamics between microbial communities and host immune responses shape immune maturation and homeostasis, influencing health outcomes. We evaluated the hypothesis that the respiratory virome is linked to systemic immune responses, using peripheral blood and nasopharyngeal swab samples from preschool-age children in the PreDicta cohort., Methods: Peripheral blood mononuclear cells from 51 children (32 asthmatics and 19 healthy controls) participating in the 2-year multinational PreDicta cohort were cultured with bacterial (Bacterial-DNA, LPS) or viral (R848, Poly:IC, RV) stimuli. Supernatants were analysed by Luminex for the presence of 22 relevant cytokines. Virome composition was obtained using untargeted high throughput sequencing of nasopharyngeal samples. The metagenomic data were used for the characterization of virome profiles and the presence of key viral families (Picornaviridae, Anelloviridae, Siphoviridae). These were correlated to cytokine secretion patterns, identified through hierarchical clustering and principal component analysis., Results: High spontaneous cytokine release was associated with increased presence of Prokaryotic virome profiles and reduced presence of Eukaryotic and Anellovirus profiles. Antibacterial responses did not correlate with specific viral families or virome profile; however, low antiviral responders had more Prokaryotic and less Eukaryotic virome profiles. Anelloviruses and Anellovirus-dominated profiles were equally distributed among immune response clusters. The presence of Picornaviridae and Siphoviridae was associated with low interferon-λ responses. Asthma or allergy did not modify these correlations., Conclusion: Antiviral cytokine responses at a systemic level reflect the upper airway virome composition. Individuals with low innate interferon responses have higher abundance of Picornaviruses (mostly Rhinoviruses) and bacteriophages. Bacteriophages, particularly Siphoviridae, appear to be sensitive sensors of host antimicrobial capacity, while Anelloviruses are not correlated with TLR-induced immune responses., (© 2023 The Authors. Allergy published by European Academy of Allergy and Clinical Immunology and John Wiley & Sons Ltd.)

Background: NLRP3-driven inflammatory responses by circulating and lung-resident monocytes are critical drivers of asthma pathogenesis. Autophagy restrains NLRP3-induced monocyte activation in asthma models. Yet, the effects of autophagy and its master regulator, transcription factor EB (TFEB), on monocyte responses in human asthma remain unexplored. Here, we investigated whether activation of autophagy and TFEB signaling suppress inflammatory monocyte responses in asthmatic individuals., Methods: Peripheral blood CD14 + monocytes from asthmatic patients (n = 83) and healthy controls (n = 46) were stimulated with LPS/ATP to induce NLRP3 activation with or without the autophagy inducer, rapamycin. ASC specks, caspase-1 activation, IL-1β and IL-18 levels, mitochondrial function, ROS release, and mTORC1 signaling were examined. Autophagy was evaluated by LC3 puncta formation, p62/SQSTM1 degradation and TFEB activation. In a severe asthma (SA) model, we investigated the role of NLRP3 signaling using Nlrp3 -/- mice and/or MCC950 administration, and the effects of TFEB activation using myeloid-specific TFEB-overexpressing mice or administration of the TFEB activator, trehalose., Results: We observed increased NLRP3 inflammasome activation, concomitant with impaired autophagy in circulating monocytes that correlated with asthma severity. SA patients also exhibited mitochondrial dysfunction and ROS accumulation. Autophagy failed to inhibit NLRP3-driven monocyte responses, due to defective TFEB activation and excessive mTORC1 signaling. NLRP3 blockade restrained inflammatory cytokine release and linked airway disease. TFEB activation restored impaired autophagy, attenuated NLRP3-driven pulmonary inflammation, and ameliorated SA phenotype., Conclusions: Our studies uncover a crucial role for TFEB-mediated reprogramming of monocyte inflammatory responses, raising the prospect that this pathway can be therapeutically harnessed for the management of SA., (© 2022 European Academy of Allergy and Clinical Immunology and John Wiley & Sons Ltd.)

Background: Although FoxP3 + regulatory T (Treg) cells constitute a highly heterogeneous population, with different regulatory potential depending on the disease context, distinct subsets or phenotypes remain poorly defined. This hampers the development of immunotherapy for allergic and autoimmune disorders. The present study aimed at characterizing distinct FoxP3 + Treg subpopulations involved in the suppression of Th2-mediated allergic inflammation in the lung., Methods: We used an established mouse model of allergic airway disease based on ovalbumin sensitization and challenge to analyze FoxP3 + Tregs during the induction and resolution of inflammation, and identify markers that distinguish their most suppressive phenotypes. We also developed a new knock-in mouse model (Foxp3 cre Cd103 dtr ) enabling the specific ablation of CD103 + FoxP3 + Tregs for functional studies., Results: We found that during resolution of allergic airway inflammation in mice >50% of FoxP3 + Treg cells expressed the integrin CD103 which marks FoxP3 + Treg cells of high IL-10 production, increased expression of immunoregulatory molecules such as KLRG1, ICOS and CD127, and enhanced suppressive capacity for Th2-mediated inflammatory responses. CD103 + FoxP3 + Tregs were essential for keeping allergic inflammation under control as their specific depletion in Foxp3 cre Cd103 dtr mice lead to severe alveocapillary damage, eosinophilic pneumonia, and markedly reduced lifespan of the animals. Conversely, adoptive transfer of CD103 + FoxP3 + Tregs effectively treated disease, attenuating Th2 responses and allergic inflammation in an IL-10-dependent manner., Conclusions: Our study identifies a novel regulatory T-cell population, defined by CD103 expression, programmed to prevent exuberant type 2 inflammation and keep homeostasis in the respiratory tract under control. This has important therapeutic implications., (© 2021 European Academy of Allergy and Clinical Immunology and John Wiley & Sons Ltd.)

Objective: Mediation of RNA interference by oligonucleotides constitutes a powerful approach for the silencing of genes involved in the pathogenesis of inflammatory disease, but in vivo application of this technique requires effective delivery to immune cells and/or sites of inflammation. The aim of the present study was to develop a new carrier system to mediate systemic administration of oligonucleotides to rheumatoid arthritis (RA) joints, and to develop an antisense oligonucleotide (ASO)-based approach to interfere with CD40-CD154 interactions in an experimental model of RA., Methods: A novel liposomal carrier with amphoteric properties, termed Nov038, was developed and assessed for its ability to systemically deliver an ASO directed against CD40 (CD40-ASO). Male DBA/1 mice with collagen-induced arthritis were treated with Nov038-encapsulated CD40-ASO, and the effects of treatment on various parameters of disease activity, including clinical score, paw swelling, lymph node responses, and inflammatory cytokine production in the joints, were assessed., Results: Nov038 was well tolerated, devoid of immune-stimulatory effects, and efficacious in mediating systemic oligonucleotide delivery to sites of inflammation. In mice with collagen-induced arthritis, Nov038 enabled the therapeutic administration of CD40-ASO and improved established disease, while unassisted CD40-ASO was ineffective, and anti-tumor necrosis factor alpha (anti-TNFalpha) treatment was less effective in this model. Nov038/CD40-ASO efficacy was attributed to its tropism for monocyte/macrophages and myeloid dendritic cells (DCs), resulting in rapid down-regulation of CD40, inhibition of DC antigen presentation, and reduction in collagen-specific T cell responses, as well as decreased levels of TNFalpha, interleukin-6 (IL-6), and IL-17 in arthritic joints., Conclusion: Amphoteric liposomes represent a novel carrier concept for systemic and antigen-presenting cell-targeted oligonucleotide delivery with clinical applicability and numerous potential applications, including target validation in vivo and inflammatory disease therapeutics. Moreover, Nov038/CD40-ASO constitutes a potent alternative to monoclonal antibody-based approaches for interfering with CD40-CD40L interactions.

Objective: To investigate the potential role of IkappaB kinase 1 (IKK-1) and IKK-2 in the regulation of nuclear factor kappaB (NF-kappaB) activation and the expression of tumor necrosis factor alpha (TNFalpha), as well as interleukin-1beta (IL-1beta), IL-6, IL-8, vascular endothelial growth factor (VEGF), and matrix metalloproteinases (MMPs), in rheumatoid arthritis (RA)., Methods: Recombinant adenoviruses expressing beta-galactosidase, dominant-negative IKK-1 and IKK-2, or IkappaBalpha were used to infect ex vivo RA synovial membrane cultures and synovial fibroblasts obtained from patients with RA undergoing joint replacement surgery, or human dermal fibroblasts, human umbilical vein endothelial cells (HUVECs), and monocyte-derived macrophages from healthy volunteers. Then, their effect on the spontaneous or stimulus-induced release of inflammatory cytokines, VEGF, and MMPs from RA synovial membrane cells was examined., Results: IKK-2 was not required for lipopolysaccharide (LPS)-induced NF-kappaB activation or TNFalpha, IL-6, or IL-8 production in macrophages, but was essential for this process in response to CD40 ligand, TNFalpha, and IL-1. In synovial fibroblasts, dermal fibroblasts, and HUVECs, IKK-2 was also required for LPS-induced NF-kappaB activation and IL-6 or IL-8 production. In RA synovial membrane cells, IKK-2 inhibition had no effect on spontaneous TNFalpha production but significantly reduced IL-1beta, IL-6, IL-8, VEGF, and MMPs 1, 2, 3, and 13., Conclusion: Our study demonstrates that IKK-2 is not essential for TNFalpha production in RA. However, because IKK-2 regulates the expression of other inflammatory cytokines (IL-1beta, IL-6, and IL-8), VEGF, and MMPs 1, 2, 3, and 13, which are involved in the inflammatory, angiogenic, and destructive processes in the RA joint, it may still be a good therapeutic target.