Activity of the NLRP3 inflammasome, a critical mediator of inflammation, is controlled by accessory proteins, posttranslational modifications, cellular localization, and oligomerization. How these factors relate is unclear. We show that a well-established drug target, Bruton’s tyrosine kinase (BTK), affects several levels of NLRP3 regulation. BTK directly interacts with NLRP3 in immune cells and phosphorylates four conserved tyrosine residues upon inflammasome activation, in vitro and in vivo. Furthermore, BTK promotes NLRP3 relocalization, oligomerization, ASC polymerization, and full inflammasome assembly, probably by charge neutralization, upon modification of a polybasic linker known to direct NLRP3 Golgi association and inflammasome nucleation. As NLRP3 tyrosine modification by BTK also positively regulates IL-1β release, we propose BTK as a multifunctional positive regulator of NLRP3 regulation and BTK phosphorylation of NLRP3 as a novel and therapeutically tractable step in the control of inflammation.
Neutrophil extracellular traps (NETs) have emerged as a key feature of cellular innate immunity mediated by polymorphonuclear neutrophils (PMNs), the primary leukocyte population in humans. Forming web-like structures composed of DNA, histones, and antimicrobial proteins, NETs trap and kill microbial invaders and thus enhance host defense. However, they have also been linked to inflammatory states, e.g. in atherosclerosis or psoriasis. Whilst DNA has been in focus as a primary structural component of NETs, we here characterize naRNA (NET-associated RNA), as a new canonical, abundant, and largely unexplored NET component. naRNA decorated all types of NETs in complex with the antimicrobial peptide LL37. In fact, naRNA was pre-associated with LL37 intracellularly as a "composite" danger-associated molecular pattern (DAMP) prior to neutrophil activation. Externalized, naRNA propagated NET formation in naive PMN, dependent on TLR8 in humans and Tlr13 in mice, in vitro and in vivo. naRNA-TLR8/Tlr13 signaling contributed significantly to the highly sensitive pro-inflammatory response of both tissue cells, like keratinocytes, and other immune cell types, such as macrophages. Those responses could be blocked by inhibition and genetic ablation of RNA receptors or RNase treatment. Importantly, in vivo naRNA strongly drove skin inflammation whereas genetic ablation of RNA sensing drastically ameliorated skin inflammation in the imiquimod psoriasis model. Our data highlight naRNA as a novel composite DAMP signaling and amplifying neutrophil activation. Moreover, naRNA emerges as the likely driver of inflammation in conditions previously linked to NETs and extracellular RNA, suggesting blockade of TLR-mediated RNA sensing as potential new intervention target
Antitumor antibodies like Rituximab and Trastuzumab activate the patient’s immune system to engage malignant cells. Despite the advances achieved by their introduction in cancer treatment, an urgent medical need remains to improve and optimize the efficacy of antibody therapy. This may be achievable using the cytokine IL-15 which induces proliferation and activation of both, NK cells and CD8 T cells, and enhances their cytolytic capacity. So far, short half-life, poor accumulation at the tumor site and severe toxicity upon systemic application due to unspecific immune activation prevent the broad use of IL-15 for treatment of cancer patients (Conlon et al, JCO 2015). IL-15 predominantly acts as part of an immunological synapse with IL-15Rα expressed on antigen-presenting cells, which present IL-15 in trans to IL-15Rβγ receptor expressing cytotoxic lymphocytes. We here report on the generation of so called “modified immunocytokines” (MICs) which consist of an Fc-optimized (SDIE modification) CD19 or CD20 antibody for targeting of (malignant) B cells fused to an IL-15 mutant with abolished binding to IL-15Rα termed MIC19 or MIC20. The abrogation of IL-15Rα binding by the IL-15 mutation enables substitution of the physiological trans-presentation by target binding of the antibody, allowing for target antigen-restricted stimulation of the IL-15Rβγ on NK cells and CD8 T cells. Analyses of activation and toxicity with MIC19 compared to a CD19 immunocytokine with an unmodified IL-15 (IC19) or immunocytokines with unrelated specificity as control confirmed target antigen-restricted function of the MIC proteins. Killing of autologous and allogeneic CD19+/CD20+ target cells was clearly superior as compared to the CD19-directed Fc optimized antibody (Tafasitamab) and Rituximab that bear no cytokine activity. Compared to antibody treatment, the MIC proteins induced a pronounced and long-lasting proliferation of effector cells and were effective to treat leukemia in a xenograft (Nalm-6) leukemia mouse model. Anti-leukemic activity was further confirmed in a murine NSG xenotransplantation model using patient ALL cells and human NK cells as effectors. In summary, MIC constructs consisting of an Fc-optimized antibody and mutated IL-15 with deficient IL-15Rα binding induce target cell restricted activation and proliferation of NK cells, resulting in efficient anti-leukemic activity. Our MICs thus combine the benefits of Fc-optimized antibodies and IL-15 activity and constitute a promising novel treatment modality for B cell malignancies with low side effects that allow the application of truly effective doses. Citation Format: Ilona Hagelstein, Latifa Zekri, Boris Klimovich, Martina S. Lutz, Carsten Greve, Stefanie Müller, Melanie Märklin, Bastian Schmied, Gundram Jung, Helmut R. Salih. Immunocytokines with target cell-restricted IL-15 activity for treatment of B cell malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2863.
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