Stromal-driven and Amyloid β-dependent induction of neutrophil extracellular traps modulates tumor growth

Abstract: Tumors consist of cancer cells and a network of non-cancerous stroma. Cancer-associated fibroblasts (CAF) are known to support tumorigenesis, and are emerging as immune modulators. Neutrophils release histone-bound nuclear DNA and cytotoxic granules as extracellular traps (NET). Here we show that CAFs induce NET formation within the tumor and systemically in the blood and bone marrow. These tumor-induced NETs (t-NETs) are driven by a ROS-mediated pathway dependent on CAF-derived Amyloid β, a peptide implicated in both neurodegenerative and inflammatory disorders. Inhibition of NETosis in murine tumors skews neutrophils to an anti-tumor phenotype, preventing tumor growth; reciprocally, t-NETs enhance CAF activation. Mirroring observations in mice, CAFs are detected juxtaposed to NETs in human melanoma and pancreatic adenocarcinoma, and show elevated amyloid and β-Secretase expression which correlates with poor prognosis. In summary, we report that CAFs drive NETosis to support cancer progression, identifying Amyloid β as the protagonist and potential therapeutic target

Reset of inflammatory priming of joint tissue and reduction of the severity of arthritis flares by bromodomain inhibition

OBJECTIVE: We have recently shown that priming of synovial fibroblasts (SFs) drives arthritis flares. Pathogenic priming of SFs is essentially mediated by epigenetic reprogramming. Bromodomain and extra-terminal motif (BET) proteins translate epigenetic changes into transcription. Here we used a BET inhibitor to target inflammatory tissue priming and reduce flare severity in experimental arthritis. METHODS: BALB/c mice were treated intraperitoneally or locally into the paw with I-BET151, which blocks interaction of BET proteins with acetylated histones. Effect of I-BET151 on acute arthritis and/or inflammatory tissue priming was assessed in a model of repeated injections of monosodium urate crystals or zymosan into the paw. I-BET151 was given either from before arthritis induction, at peak inflammation, or after healing of the first arthritis bout. Transcriptomic (RNA-Seq), epigenomic (ATAC-Seq) and functional analysis (invasion, cytokine production, migration, senescence, metabolic flux) was performed on murine and human SFs treated with I-BET151 in vitro or in vivo. RESULTS: Systemic I-BET151 administration did not affect acute inflammation but abolished inflammatory tissue priming and diminished flare severity in both preventive and therapeutic treatment settings. I-BET151 was also effective when applied locally in the joint. BET inhibition also inhibited osteoclast differentiation, while macrophage activation in the joint was not affected. Flare reduction after BET inhibition was mediated, at least in part, by rolling back the primed transcriptional, metabolic and pathogenic phenotype of SFs. CONCLUSION: Inflammatory tissue priming is dependent on transcriptional regulation by BET proteins, which makes them promising therapeutic targets for preventing arthritis flares in previously affected joints

Neutrophils’ Extracellular Trap Mechanisms: From Physiology to Pathology

Neutrophils are an essential part of the innate immune system and the first line of defense against invading pathogens. They phagocytose, release granular contents, produce reactive oxygen species, and form neutrophil extracellular traps (NETs) to fight pathogens. With the characterization of NETs and their components, neutrophils were identified as players of the innate adaptive crosstalk. This has placed NETs at the center not only of physiological but also pathological processes. Aside from their role in pathogen uptake and clearance, NETs have been demonstrated to contribute to the resolution of inflammation by forming aggregated NETs able to degrade inflammatory mediators. On the other hand, NETs have the potential to foster severe pathological conditions. When homeostasis is disrupted, they occlude vessels and ducts, serve as sources of autoantigens and danger or damage associated molecular patterns, directly damage tissues, and exaggerate complement activity and inflammation. This review focusses on the understanding of NETs from their formation to their functions in both physiological and pathological processes

Stunning of neutrophils accounts for the anti-inflammatory effects of clodronate liposomes.

Clodronate liposomes (Clo-Lip) have been widely used to deplete mononuclear phagocytes (MoPh) to study the function of these cells in vivo. Here, we revisited the effects of Clo-Lip together with genetic models of MoPh deficiency, revealing that Clo-Lip exert their anti-inflammatory effects independent of MoPh. Notably, not only MoPh but also polymorphonuclear neutrophils (PMN) ingested Clo-Lip in vivo, which resulted in their functional arrest. Adoptive transfer of PMN, but not of MoPh, reversed the anti-inflammatory effects of Clo-Lip treatment, indicating that stunning of PMN rather than depletion of MoPh accounts for the anti-inflammatory effects of Clo-Lip in vivo. Our data highlight the need for a critical revision of the current literature on the role of MoPh in inflammation.This work was supported by the Deutsche Forschungsgemeinschaft (FG 2886 “PANDORA” – B01/B02/A03/ B04 to G. Kronke, F. Nimmerjahn, G. Schett, and M.H. Hoff- ¨ mann, respectively, and the CRC1181-A03/A01/A02/A07/C03 Z2 to G. Kronke, G. Schett, F. Nimmerjahn, and M.H. Hoffmann), ¨ the Emerging Field Initiative of the Friedrich-Alexander University Erlangen-Nürnberg (EFI_Verbund_Med_05_MIRACLE to G. Kronke), the Bundesministerium für Bildung und For- ¨ schung (MASCARA to G. Kronke and G. Schett; MelAutim to G. ¨ Kronke), and the European Union (Horizon 2020 ERC-2014-StG ¨ 640087 – SOS and ERC-2020-CoG 101001866 – INSPIRE to G. Kronke; and ERC-2018-SyG nanoSCOPE and RTCure to G. ¨ Schett). This work was supported by grants R01AI165661 from the National Institutes of Health/National Institute of Allergy and Infectious Diseases, H2020-FET-OPEN-2018- 2020 (no. 861878) from the European Commission to A. Hidalgo and M.H. Hoffmann, and HR17_00527 from Fundacion La Caixa to A. Hidalgo. The CNIC is supported by the Ministerio de Ciencia e Innovacion and the Pro-CNIC Foundation and is a Severo Ochoa Center of Excellence (MICINN award CEX2020-001041-S).S

Reset of inflammatory priming of joint tissue and reduction of the severity of arthritis flares by bromodomain inhibition.

OBJECTIVE We have recently shown that priming of synovial fibroblasts (SFs) drives arthritis flares. Pathogenic priming of SFs is essentially mediated by epigenetic reprogramming. Bromodomain and extra-terminal motif (BET) proteins translate epigenetic changes into transcription. Here we used a BET inhibitor to target inflammatory tissue priming and reduce flare severity in experimental arthritis. METHODS BALB/c mice were treated intraperitoneally or locally into the paw with I-BET151, which blocks interaction of BET proteins with acetylated histones. Effect of I-BET151 on acute arthritis and/or inflammatory tissue priming was assessed in a model of repeated injections of monosodium urate crystals or zymosan into the paw. I-BET151 was given either from before arthritis induction, at peak inflammation, or after healing of the first arthritis bout. Transcriptomic (RNA-Seq), epigenomic (ATAC-Seq) and functional analysis (invasion, cytokine production, migration, senescence, metabolic flux) was performed on murine and human SFs treated with I-BET151 in vitro or in vivo. RESULTS Systemic I-BET151 administration did not affect acute inflammation but abolished inflammatory tissue priming and diminished flare severity in both preventive and therapeutic treatment settings. I-BET151 was also effective when applied locally in the joint. BET inhibition also inhibited osteoclast differentiation, while macrophage activation in the joint was not affected. Flare reduction after BET inhibition was mediated, at least in part, by rolling back the primed transcriptional, metabolic and pathogenic phenotype of SFs. CONCLUSION Inflammatory tissue priming is dependent on transcriptional regulation by BET proteins, which makes them promising therapeutic targets for preventing arthritis flares in previously affected joints

Therapeutic ACPA inhibits NET formation: a potential therapy for neutrophil-mediated inflammatory diseases

Excessive release of neutrophil extracellular traps (NETs) is associated with disease severity and contributes to tissue injury, followed by severe organ damage. Pharmacological or genetic inhibition of NET release reduces pathology in multiple inflammatory disease models, indicating that NETs are potential therapeutic targets. Here, we demonstrate using a preclinical basket approach that our therapeutic anti-citrullinated protein antibody (tACPA) has broad therapeutic potential. Treatment with tACPA prevents disease symptoms in various mouse models with plausible NET-mediated pathology, including inflammatory arthritis (IA), pulmonary fibrosis, inflammatory bowel disease and sepsis. We show that citrulline residues in the N-termini of histones 2A and 4 are specific targets for therapeutic intervention, whereas antibodies against other N-terminal post-translational histone modifications have no therapeutic effects. Because citrullinated histones are generated during NET release, we investigated the ability of tACPA to inhibit NET formation. tACPA suppressed NET release from human neutrophils triggered with physiologically relevant human disease-related stimuli. Moreover, tACPA diminished NET release and potentially initiated NET uptake by macrophages in vivo, which was associated with reduced tissue damage in the joints of a chronic arthritis mouse model of IA. To our knowledge, we are the first to describe an antibody with NET-inhibiting properties and thereby propose tACPA as a drug candidate for NET-mediated inflammatory diseases, as it eliminates the noxious triggers that lead to continued inflammation and tissue damage in a multidimensional manner

The complement system drives local inflammatory tissue priming by metabolic reprogramming of articular fibroblasts

Arthritis typically involves recurrence and progressive worsening at specific predilection sites, but the checkpoints between remission and persistence remain unknown. Here, we defined the molecular and cellular mechanisms of this inflammation-mediated tissue priming. Re-exposure to inflammatory stimuli caused aggravated arthritis in rodent models. Tissue priming developed locally and independently of adaptive immunity. Repeatedly stimulated primed synovial fibroblasts (SFs) exhibited enhanced metabolic activity inducing functional changes with intensified migration, invasiveness and osteoclastogenesis. Meanwhile, human SF from patients with established arthritis displayed a similar primed phenotype. Transcriptomic and epigenomic analyses as well as genetic and pharmacological targeting demonstrated that inflammatory tissue priming relies on intracellular complement C3- and C3a receptor-activation and downstream mammalian target of rapamycin- and hypoxia-inducible factor 1α-mediated metabolic SF invigoration that prevents activation-induced senescence, enhances NLRP3 inflammasome activity, and in consequence sensitizes tissue for inflammation. Our study suggests possibilities for therapeutic intervention abrogating tissue priming without immunosuppression

The complement system drives local inflammatory tissue priming by metabolic reprogramming of synovial fibroblasts.

Arthritis typically involves recurrence and progressive worsening at specific predilection sites, but the checkpoints between remission and persistence remain unknown. Here, we defined the molecular and cellular mechanisms of this inflammation-mediated tissue priming. Re-exposure to inflammatory stimuli caused aggravated arthritis in rodent models. Tissue priming developed locally and independently of adaptive immunity. Repeatedly stimulated primed synovial fibroblasts (SFs) exhibited enhanced metabolic activity inducing functional changes with intensified migration, invasiveness and osteoclastogenesis. Meanwhile, human SF from patients with established arthritis displayed a similar primed phenotype. Transcriptomic and epigenomic analyses as well as genetic and pharmacological targeting demonstrated that inflammatory tissue priming relies on intracellular complement C3- and C3a receptor-activation and downstream mammalian target of rapamycin- and hypoxia-inducible factor 1α-mediated metabolic SF invigoration that prevents activation-induced senescence, enhances NLRP3 inflammasome activity, and in consequence sensitizes tissue for inflammation. Our study suggests possibilities for therapeutic intervention abrogating tissue priming without immunosuppression