Search

The coagulation cascade and fibrinolysis have links with neuroinflammation and increased activation of the coagulation system has been reported in MS patients. We quantified levels of D-dimer, tissue plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1) and the bioactivity of bacterial lipopolysaccharide (LPS) in cerebrospinal fluid (CSF) and plasma from newly diagnosed untreated MS patients and controls. These molecules showed multiple correlations with each other as well as with age, HLA-DRB1*15:01, body-mass-index and CSF IgG. Our results confirm previous findings of increased plasma PAI-1 and LPS in MS patients compared to controls indicating changes in platelet function and gut permeability in MS., Competing Interests: Declaration of competing interest JL: Congress expenses, Merck. PJT: Lecture and consulting fees Roche, Merck, Biogen, Novartis, Janssen, Sanofi, Alexion; congress expenses Biogen, Merck. KKE: Lecture fees: Novartis, Celltrion, Sobi., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Background and Objectives: Epstein-Barr virus (EBV) infection is a major risk factor of multiple sclerosis (MS). We examined the presence of EBV DNA in the CSF and blood of patients with MS and controls. We analyzed whether EBV DNA is more common in the CSF of patients with MS than in controls and estimated the proportions of EBV-positive B cells in the CSF and blood., Methods: CSF supernatants and cells were collected at diagnostic lumbar punctures from 45 patients with MS and 45 HLA-DR15 matched controls with other conditions, all participants were EBV seropositive. Cellular DNA was amplified by Phi polymerase targeting both host and viral DNA, and representative samples were obtained in 28 cases and 28 controls. Nonamplified DNA from CSF cells (14 cases, 14 controls) and blood B cells (10 cases, 10 controls) were analyzed in a subset of participants. Multiple droplet digital PCR (ddPCR) runs were performed per sample to assess the cumulative EBV positivity rate. To detect viral RNA as a sign of activation, RNA sequencing was performed in blood CD4-positive, CD8-positive, and CD19-positive cells from 21 patients with MS and 3 controls., Results: One of the 45 patients with MS and none of the 45 controls were positive for EBV DNA in CSF supernatants (1 mL). CSF cellular DNA was analyzed in 8 independent ddPCRs: EBV DNA was detected at least once in 18 (64%) of the 28 patients with MS and in 15 (54%) of the 28 controls ( p = 0.59, Fisher test). The cumulative EBV positivity increased steadily up to 59% in the successive ddPCRs, suggesting that all individuals would have reached EBV positivity in the CSF cells, if more DNA would have been analyzed. The estimated proportion of EBV-positive B cells was >1/10,000 in both the CSF and blood. We did not detect viral RNA, except from endogenous retroviruses, in the blood lymphocyte subpopulations., Discussion: EBV-DNA is equally detectable in the CSF cells of both patients with MS and controls with ddPCR, and the probabilistic approach indicates that the true positivity rate approaches 100% in EBV-positive individuals. The proportion of EBV-positive B cells seems higher than previously estimated.

In monogenic autoinflammatory diseases, mutations in genes regulating innate immune responses often lead to uncontrolled activation of inflammasome pathways or the type I interferon (IFN-I) response. We describe a mechanism of autoinflammation potentially predisposing patients to life-threatening necrotizing soft tissue inflammation. Six unrelated families are identified in which affected members present with necrotizing fasciitis or severe soft tissue inflammations. Exome sequencing reveals truncating monoallelic loss-of-function variants of nuclear factor κ light-chain enhancer of activated B cells (NFKB1) in affected patients. In patients' macrophages and in NFKB1-variant-bearing THP-1 cells, activation increases both interleukin (IL)-1β secretion and IFN-I signaling. Truncation of NF-κB1 impairs autophagy, accompanied by the accumulation of reactive oxygen species and reduced degradation of inflammasome receptor nucleotide-binding oligomerization domain, leucine-rich repeat-containing protein 3 (NLRP3), and Toll/IL-1 receptor domain-containing adaptor protein inducing IFN-β (TRIF), thus leading to combined excessive inflammasome and IFN-I activity. Many of the patients respond to anti-inflammatory treatment, and targeting IL-1β and/or IFN-I signaling could represent a therapeutic approach for these patients., Competing Interests: Declaration of interests C.J.A.D. has provided consultative advice to Synairgen on behalf of Newcastle University., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Immune-mediated arthritis is a group of autoinflammatory diseases, where the patient's own immune system attacks and destroys synovial joints. Sustained remission is not always achieved with available immunosuppressive treatments, warranting more detailed studies of T cell responses that perpetuate synovial inflammation in treatment-refractory patients., Methods: In this study, we investigated CD4 + and CD8 + T lymphocytes from the synovial tissue and peripheral blood of patients with treatment-resistant immune-mediated arthritis using paired single-cell RNA and TCR-sequencing. To gain insights into the trafficking of clonal families, we compared the phenotypes of clones with the exact same TCRß amino acid sequence between the two tissues., Results: Our results show that both CD4 + and CD8 + T cells display a more activated and inflamed phenotype in the synovial tissue compared to peripheral blood both at the population level and within individual T cell families. Furthermore, we found that both cell subtypes exhibited clonal expansion in the synovial tissue., Conclusions: Our findings suggest that the local environment in the synovium drives the proliferation of activated cytotoxic T cells, and both CD4 + and CD8 + T cells may contribute to tissue destruction and disease pathogenesis., (© 2024. The Author(s).)

Loss-of-function (LOF) mutations in NFKB1, coding for p105, may cause common variable immunodeficiency due to dysregulation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κΒ) pathway. Monoallelic LOF variants of NFKB1 can predispose to uncontrolled inflammation including sterile necrotizing fasciitis or pyoderma gangrenosum. In this study, we explored the impact of a heterozygous NFKB1 c.C936T/p.R157X LOF variant on immunity in sterile fasciitis patients and their family members. The p50 or p105 protein levels were reduced in all variant carriers. Interleukin-1β (IL-1β) and interleukin-8 (IL-8) levels were elevated in vitro, potentially contributing to the very high neutrophil counts observed during fasciitis episodes. Phosphorylation of p65/RelA was reduced in p.R157X neutrophils suggesting defective activation of canonical NF-κB. Oxidative burst after NF-κB-independent phorbol 12-myristate 13-acetate (PMA) stimulation was similar in both p.R157X and control neutrophils. Comparable amounts of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex subunits were found in p.R157X and control neutrophils. However, a compromised oxidative burst was observed in p.R157X neutrophils following activation of NF-κB-dependent mechanisms following stimulation of toll-like receptor 2 (TLR2) and Dectin-1. Neutrophil extracellular trap formation was not affected by p.R157X. In summary, the NFKB1 c.C936T/p.R157X LOF variant has an impact on inflammation and neutrophil function and may play a role in the pathogenesis of sterile necrotizing fasciitis., (© 2023. The Author(s).)

Hairpin oligodeoxynucleotides incorporating a (2R,3S)-4-(methoxyamino)butane-1,2,3-triol residue in the middle of the double-helical stem and opposite to either one of the canonical nucleobases or an abasic 2-(hydroxymethyl)tetrahydrofuran-3-ol spacer were synthesized. Under mildly acidic conditions, aromatic aldehydes reacted reversibly with these oligonucleotides, converting the (2R,3S)-4-(methoxyamino)butane-1,2,3-triol unit into a 2-aryl-N-methoxy-1,3-oxazinane nucleoside analogue. The equilibrium of this reaction was found to be dependent on both the aldehyde and the nucleobase opposite to the modified residue. 9-Formyl-9-deazaadenine, combining a large stacking surface with an array of hydrogen bond donors and acceptors, showed the highest affinity as well as selectivity consistent with the rules of Watson-Crick base pairing. 5-Formyluracil or indole-3-carbaldehyde, lacking in either stacking or hydrogen bonding ability, were incorporated with a much lower affinity and selectivity., (© 2023The Authors. Published by Wiley-VCH GmbH.)

Objective: IL-17A and TNF act in synergy to induce proinflammatory mediators in synovial fibroblasts thus contributing to diseases associated with chronic arthritis. Many of these factors are regulated by transcription factor E74-like factor-3 (ELF3). Therefore, we sought to investigate ELF3 as a downstream target of IL-17A and TNF signalling and to characterize its role in the molecular mechanism of synergy between IL-17A and TNF., Methods: Regulation of ELF3 expression by IL-17A and TNF was studied in synovial fibroblasts of RA and OA patients and RA synovial explants. Signalling leading to ELF3 mRNA induction and the impact of ELF3 on the response to IL-17A and TNF were studied using siRNA, transient overexpression and signalling inhibitors in synovial fibroblasts and HEK293 cells., Results: ELF3 was marginally affected by IL-17A or TNF alone, but their combination resulted in high and sustained expression. ELF3 expression was regulated by the nuclear factor-κB (NF-κB) pathway and CCAAT/enhancer-binding protein β (C/EBPβ), but its induction required synthesis of the NF-κB co-factor IκB (inhibitor of NF-κB) ζ. siRNA-mediated depletion of ELF3 attenuated the induction of cytokines and matrix metalloproteinases by the combination of IL-17A and TNF. Overexpression of ELF3 or IκBζ showed synergistic effect with TNF in upregulating expression of chemokine (C-C motif) ligand 8 (CCL8), and depletion of ELF3 abrogated CCL8 mRNA induction by the combination of IκBζ overexpression and TNF., Conclusion: Altogether, our results establish ELF3 as an important mediator of the synergistic effect of IL-17A and TNF in synovial fibroblasts. The findings provide novel information of the pathogenic mechanisms of IL-17A in chronic arthritis and implicate ELF3 as a potential therapeutic target., (© The Author(s) 2022. Published by Oxford University Press on behalf of the British Society for Rheumatology.)

Human macrophages secrete extracellular vesicles (EVs) loaded with numerous immunoregulatory proteins. Vesicle-mediated protein secretion in macrophages is regulated by poorly characterized mechanisms; however, it is now known that inflammatory conditions significantly alter both the quantities and protein composition of secreted vesicles. In this study, we employed high-throughput quantitative proteomics to characterize the modulation of EV-mediated protein secretion during noncanonical caspase-4/5 inflammasome activation via LPS transfection. We show that human macrophages activate robust caspase-4-dependent EV secretion upon transfection of LPS, and this process is also partially dependent on NLRP3 and caspase-5. A similar effect occurs with delivery of the LPS with Escherichia coli-derived outer membrane vesicles. Moreover, sensitization of the macrophages through TLR4 by LPS priming prior to LPS transfection dramatically augments the EV-mediated protein secretion. Our data demonstrate that this process differs significantly from canonical inflammasome activator ATP-induced vesiculation, and it is dependent on the autocrine IFN signal associated with TLR4 activation. LPS priming preceding the noncanonical inflammasome activation significantly enhances vesicle-mediated secretion of inflammasome components caspase-1, ASC, and lytic cell death effectors GSDMD, MLKL, and NINJ1, suggesting that inflammatory EV transfer may exert paracrine effects in recipient cells. Moreover, using bioinformatics methods, we identify 15-deoxy-Δ12,14-PGJ2 and parthenolide as inhibitors of caspase-4-mediated inflammation and vesicle secretion, indicating new therapeutic potential of these anti-inflammatory drugs., (Copyright © 2023 by The American Association of Immunologists, Inc.)

Background: Dysbiotic intestinal and oral microbiota have been implicated in the pathogenesis of rheumatoid arthritis (RA), but the mechanisms how microbiota could impact disease activity have remained elusive. The aim of this study was to assess the association of the biological activity of serum lipopolysaccharides (LPS) with disease activity and likelihood of achieving remission in RA patients., Methods: We measured Toll-like receptor (TLR) 4-stimulating activity of sera of 58 RA patients with a reporter cell line engineered to produce secreted alkaline phosphatase in response to TLR4 stimulation. Levels of LPS-binding protein, CD14, and CD163 were determined by ELISA assays., Results: The patient serum-induced TLR4 activation (biological activity of LPS) was significantly associated with inflammatory parameters and body mass index at baseline and at 12 months and with disease activity (DAS28-CRP, p<0.001) at 12 months. Importantly, baseline LPS bioactivity correlated with disease activity (p=0.031) and, in 28 early RA patients, the likelihood of achieving remission at 12 months (p=0.009). The level of LPS bioactivity was similar at baseline and 12-month visits, suggesting that LPS bioactivity is an independent patient-related factor. Neutralization of LPS in serum by polymyxin B abrogated the TLR4 signaling, suggesting that LPS was the major contributor to TLR4 activation., Conclusion: We describe a novel approach to study the biological activity of serum LPS and their impact in diseases. The results suggest that LPS contribute to the inflammatory burden and disease activity on patients with RA and that serum-induced TLR4 activation assays can serve as an independent prognostic factor. A graphical summary of the conclusions of the study., (© 2022. The Author(s).)

Nanostructures based on DNA self-assembly present an innovative way to address the increasing need for target-specific delivery of therapeutic molecules. Currently, most of the chemotherapeutics being used in clinical practice have undesired and exceedingly high off-target toxicity. This is a challenge in particular for small molecules, and hence, developing robust and effective methods to lower these side effects and enhance the antitumor activity is of paramount importance. Prospectively, these issues could be tackled with the help of DNA nanotechnology, which provides a route for the fabrication of custom, biocompatible, and multimodal structures, which can, to some extent, resist nuclease degradation and survive in the cellular environment. Similar to widely employed liposomal products, the DNA nanostructures (DNs) are loaded with selected drugs, and then by employing a specific stimulus, the payload can be released at its target region. This review explores several strategies and triggers to achieve targeted delivery of DNs. Notably, different modalities are explained through which DNs can interact with their respective targets as well as how structural changes triggered by external stimuli can be used to achieve the display or release of the cargo. Furthermore, the prospects and challenges of this technology are highlighted., (© 2021 Wiley-VCH GmbH.)

The Ikaros family transcription factors regulate lymphocyte development. Loss-of-function variants in IKZF1 cause primary immunodeficiency, but Ikaros family members IKZF2 and IKZF3 have not yet been associated with immunodeficiency. Here, we describe a pedigree with a heterozygous truncating variant in IKZF2 , encoding the transcriptional activator and repressor Helios, which is highly expressed in regulatory T cells and effector T cells, particularly of the CD8 + T cell lineage. Protein-protein interaction analysis revealed that the variant abolished heterodimerization of Helios with Ikaros and Aiolos and also prevented Helios binding to members of the Mi-2/NuRD chromatin remodeling complex. Patients carrying the IKZF2 variant presented with a combined immunodeficiency phenotype characterized by recurrent upper respiratory infections, thrush and mucosal ulcers, and chronic lymphadenopathy. With extensive immunophenotyping, functional assays, and transcriptional analysis, we show that reduced Helios expression was associated with chronic T cell activation and increased production of proinflammatory cytokines both in effector and regulatory T cells. Lymph node histology from patients indicated dysregulated germinal center reactions. Moreover, affected individuals displayed a profound reduction in circulating MAIT cell numbers. In summary, we show that this previously undescribed loss-of-function variant in Helios leads to an immunodeficiency with signs of immune overactivation.

Objectives: To determine the anti-inflammatory effect and safety of hydroxychloroquine after acute myocardial infarction., Method: In this multicenter, double-blind, placebo-controlled OXI trial, 125 myocardial infarction patients were randomized at a median of 43 h after hospitalization to receive hydroxychloroquine 300 mg (n = 64) or placebo (n = 61) once daily for 6 months and, followed for an average of 32 months. Laboratory values were measured at baseline, 1, 6, and 12 months., Results: The levels of interleukin-6 (IL-6) were comparable at baseline between study groups (p = 0.18). At six months, the IL-6 levels were lower in the hydroxychloroquine group (p = 0.042, between groups), and in the on-treatment analysis, the difference at this time point was even more pronounced (p = 0.019, respectively). The high-sensitivity C-reactive protein levels did not differ significantly between study groups at any time points. Eleven patients in the hydroxychloroquine group and four in the placebo group had adverse events leading to interruption or withdrawal of study medication, none of which was serious (p = 0.10, between groups)., Conclusions: In patients with myocardial infarction, hydroxychloroquine reduced IL-6 levels significantly more than did placebo without causing any clinically significant adverse events. A larger randomized clinical trial is warranted to prove the potential ability of hydroxychloroquine to reduce cardiovascular endpoints after myocardial infarction., Competing Interests: Declaration of Competing Interest JS reports personal fees from Abbott, Amgen, and Bayer and a grant from AstraZeneca. PTK reports personal fees from Amgen, Novartis, Raisio Group, and Sanofi., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Objectives: The NLRP3 inflammasome plays a key role in arterial wall inflammation. In this study, we elucidated the role of serum lipoproteins in the regulation of NLRP3 inflammasome activation by serum amyloid A (SAA) and other inflammasome activators., Methods: The effect of lipoproteins on the NLRP3 inflammasome activation was studied in primary human macrophages and THP-1 macrophages. The effect of oxidised low-density lipoprotein (LDL) was examined in an in vivo mouse model of SAA-induced peritoneal inflammation., Results: Native and oxidised high-density lipoproteins (HDL 3 ) and LDLs inhibited the interaction of SAA with TLR4. HDL 3 and LDL inhibited the secretion of interleukin (IL)-1β and tumor necrosis factor by reducing their transcription. Oxidised forms of these lipoproteins reduced the secretion of mature IL-1β also by inhibiting the activation of NLRP3 inflammasome induced by SAA, ATP, nigericin and monosodium urate crystals. Specifically, oxidised LDL was found to inhibit the inflammasome complex formation. No cellular uptake of lipoproteins was required, nor intact lipoprotein particles for the inhibitory effect, as the lipid fraction of oxidised LDL was sufficient. The inhibition of NLRP3 inflammasome activation by oxidised LDL was partially dependent on autophagy. Finally, oxidised LDL inhibited the SAA-induced peritoneal inflammation and IL-1β secretion in vivo ., Conclusions: These findings reveal that both HDL 3 and LDL inhibit the proinflammatory activity of SAA and this inhibition is further enhanced by lipoprotein oxidation. Thus, lipoproteins possess major anti-inflammatory functions that hinder the NLRP3 inflammasome-activating signals, particularly those exerted by SAA, which has important implications in the pathogenesis of cardiovascular diseases., Competing Interests: The authors declare no conflicts of interest., (© 2021 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology Inc.)

NLRP3 inflammasome has been implicated in impaired post-injury muscle healing and in muscle atrophy. Histamine receptors play an important role in inflammation, but the role of histamine H 3 receptor (H 3 R) in myocyte regeneration and in the regulation of NLRP3 inflammasome is not known. We studied the effects of H 3 R signaling on C2C12 myocyte viability, apoptosis, and tumor necrosis factor alpha (TNFα)-induced NLRP3 inflammasome activation during striated myogenic differentiation at three time points (days 0, 3, and 6). Expression of Nlrp3 , interleukin-1β (IL-1β), and myogenesis markers were determined. TNFα reduced overall viability of C2C12 cells, and exposure to TNFα induced apoptosis of cells at D6. Activation of H 3 R had no effect on viability or apoptosis, whereas inhibition of H 3 R increased TNFα-induced apoptosis. Stimulation of C2C12 cells with TNFα increased Nlrp3 mRNA expression at D3 and D6. Moreover, TNFα reduced the expression of myogenesis markers MyoD1, Myogenin, and Myosin-2 at D3 and D6. H 3 R attenuated TNFα-induced expression of Nlrp3 and further inhibited the myogenesis marker expression; while H 3 R -blockage enhanced the proinflammatory effects of TNFα and increased the myogenesis marker expression. TNFα-induced secretion of mature IL-1β was dependent on the activation of the NLRP3 inflammasome, as shown by the reduced secretion of mature IL-1β upon treatment of the cells with the small molecule inhibitor of the NLRP3 inflammasome (MCC950). The activation of H 3 R reduced TNFα-induced IL-1β secretion, while the H 3 R blockage had an opposite effect. In conclusion, the modulation of H 3 R activity regulates the effects of TNFα on C2C12 myocyte differentiation and TNFα-induced activation of NLRP3 inflammasome. Thus, H 3 R signaling may represent a novel target for limiting postinjury muscle inflammation and muscle atrophy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Chen, Ma, Feng, Wang, Li, Nurmi, Eklund and Wen.)

Aseptic loosening of total joint replacements is driven by a macrophage-mediated inflammatory reaction to implant-derived wear particles. Phagocytosis of implant debris has been suggested to activate the NLRP3 inflammasome leading to secretion of interleukin (IL)-1β. However, factors and molecular mechanisms driving the particle-induced inflammasome activation are yet to be fully elucidated. In this study, we investigated the inflammasome response of human primary macrophages to titanium, chromium, and molybdenum particles in vitro. We observed that particles alone were not sufficient to induce IL-1β secretion, but an additional priming signal-such as bacterial lipopolysaccharide (LPS)-was required to license the inflammasome activation. By using specific inhibitors against the inflammasome signaling pathway, we demonstrate that the particle-induced IL-1β secretion depended upon activation of the NLRP3 inflammasome. We further hypothesized that tumor necrosis factor (TNF) could substitute for LPS as a priming signal, and found that particle stimulation together with preceding TNF treatment resulted in inflammasome-dependent IL-1β production as well. Our results show that the NLRP3 inflammasome mediates wear particle responses in human primary macrophages, and its activation does not necessarily require the presence of bacterial components, but can be induced under aseptic conditions by TNF priming. STATEMENT OF SIGNIFICANCE: This study was conducted to elucidate the molecular mechanisms of metal particle-induced IL-1β secretion in human primary macrophages. Production of this pro-inflammatory mediator from wear particle-activated macrophages has been associated with increased bone loss around total joint replacements-a condition eventually requiring revision surgery. Our results confirm that together with a co-stimulatory priming signal, particles of common implant metals elicit macrophage-mediated IL-1β secretion through activation of the NLRP3 inflammasome pathway. We also present a concept of TNF priming in this context, demonstrating that the particle-related IL-1β secretion can take place in a truly sterile environment. Thus, inhibition of inflammasome signaling appears a means to prevent wear particle-induced inflammation and development of peri‑prosthetic osteolysis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier Ltd.)

Background: The treatment of nucleotide-binding domain and leucine-rich repeat containing family, pyrin domain containing 3 (NLRP3) inflammasome-mediated pediatric inflammatory diseases is challenging. Here we studied whether cyclic adenosine monophosphate (cAMP) elevator forskolin could attenuate the nigericin-induced NLRP3-inflammasome activation and interleukin-1β (IL-1β) secretion in human macrophages., Methods: The proteins and messenger RNA (mRNA) levels of inflammasome structural proteins and proinflammatory cytokines were measured in forskolin-stimulated nigericin-activated human THP-1 macrophages and primary macrophages., Results: Activation of THP-1 macrophages with nigericin increased the mRNA expression of NLRP3, IL-1β, and caspase-1 (P < 0.01). Forskolin stimulation had no effect on the mRNA expression of NLRP3, caspase-1, or IL-1β in nigericin-activated cells (P > 0.05), while their protein levels were significantly decreased (P < 0.05). Forskolin-mediated increase in cytoplasmic cAMP in non-activated cells was attenuated in nigericin-activated macrophages (P < 0.05). Basal IL-1β secretion increased from 584 to 2696 pg/mL (P < 0.01) in nigericin-activated macrophages; forskolin dose-dependently reduced the nigericin-induced secretion of mature IL-1β (P < 0.01). Forskolin also inhibited the IL-1β secretion from activated human primary macrophages., Conclusions: Forskolin inhibits the NLRP3 inflammasome activation and the secretion of mature IL-1β, in human macrophages. Forskolin and other cAMP elevator drugs could represent a novel approach for treatment of diseases associated with excessive inflammasome activation, like pediatric inflammatory diseases.

Background: CCAAT enhancer-binding protein epsilon (C/EBPε) is a transcription factor involved in late myeloid lineage differentiation and cellular function. The only previously known disorder linked to C/EBPε is autosomal recessive neutrophil-specific granule deficiency leading to severely impaired neutrophil function and early mortality., Objective: The aim of this study was to molecularly characterize the effects of C/EBPε transcription factor Arg219His mutation identified in a Finnish family with previously genetically uncharacterized autoinflammatory and immunodeficiency syndrome., Methods: Genetic analysis, proteomics, genome-wide transcriptional profiling by means of RNA-sequencing, chromatin immunoprecipitation (ChIP) sequencing, and assessment of the inflammasome function of primary macrophages were performed., Results: Studies revealed a novel mechanism of genome-wide gain-of-function that dysregulated transcription of 464 genes. Mechanisms involved dysregulated noncanonical inflammasome activation caused by decreased association with transcriptional repressors, leading to increased chromatin occupancy and considerable changes in transcriptional activity, including increased expression of NLR family, pyrin domain-containing 3 protein (NLRP3) and constitutively expressed caspase-5 in macrophages., Conclusion: We describe a novel autoinflammatory disease with defective neutrophil function caused by a homozygous Arg219His mutation in the transcription factor C/EBPε. Mutated C/EBPε acts as a regulator of both the inflammasome and interferome, and the Arg219His mutation causes the first human monogenic neomorphic and noncanonical inflammasomopathy/immunodeficiency. The mechanism, including widely dysregulated transcription, is likely not unique for C/EBPε. Similar multiomics approaches should also be used in studying other transcription factor-associated diseases., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: The nuclear factor κ light-chain enhancer of activated B cells (NF-κB) signaling pathway is a key regulator of immune responses. Accordingly, mutations in several NF-κB pathway genes cause immunodeficiency., Objective: We sought to identify the cause of disease in 3 unrelated Finnish kindreds with variable symptoms of immunodeficiency and autoinflammation., Methods: We applied genetic linkage analysis and next-generation sequencing and functional analyses of NFKB1 and its mutated alleles., Results: In all affected subjects we detected novel heterozygous variants in NFKB1, encoding for p50/p105. Symptoms in variant carriers differed depending on the mutation. Patients harboring a p.I553M variant presented with antibody deficiency, infection susceptibility, and multiorgan autoimmunity. Patients with a p.H67R substitution had antibody deficiency and experienced autoinflammatory episodes, including aphthae, gastrointestinal disease, febrile attacks, and small-vessel vasculitis characteristic of Behçet disease. Patients with a p.R157X stop-gain experienced hyperinflammatory responses to surgery and showed enhanced inflammasome activation. In functional analyses the p.R157X variant caused proteasome-dependent degradation of both the truncated and wild-type proteins, leading to a dramatic loss of p50/p105. The p.H67R variant reduced nuclear entry of p50 and showed decreased transcriptional activity in luciferase reporter assays. The p.I553M mutation in turn showed no change in p50 function but exhibited reduced p105 phosphorylation and stability. Affinity purification mass spectrometry also demonstrated that both missense variants led to altered protein-protein interactions., Conclusion: Our findings broaden the scope of phenotypes caused by mutations in NFKB1 and suggest that a subset of autoinflammatory diseases, such as Behçet disease, can be caused by rare monogenic variants in genes of the NF-κB pathway., (Copyright © 2017 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Inflammasomes are intracellular protein platforms, which, upon activation, produce the highly proinflammatory cytokines interleukin (IL)-1β and IL-18. Heme, hemin and their degradation products possess significant immunomodulatory functions. Here, we studied whether hemin regulates inflammasome function in macrophages. Both hemin and its derivative, cobalt protoporphyrin (CoPP), significantly reduced IL-1β secretion by cultured human primary macrophages, the human monocytic leukemia cell line and also mouse bone marrow-derived and peritoneal macrophages. Intraperitoneal administration of CoPP to mice prior to urate crystal-induced peritonitis alleviated IL-1β secretion to the peritoneal cavity. In cultured macrophages, hemin and CoPP inhibited NLRP3 inflammasome assembly by reducing the amount of intracellular apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC). The reduction of ASC was associated with enhanced autophagosome formation and autophagic flux. Inhibition of autophagy prevented the CoPP-induced depletion of ASC, implying that the depletion was caused by increased autophagy. Our data indicate that hemin functions as an endogenous negative regulator of the NLRP3 inflammasome. The inhibition is mediated via enhanced autophagy that results in increased degradation of ASC. This regulatory mechanism may provide a novel approach for the treatment of inflammasome-related diseases., (© 2016 S. Karger AG, Basel.)

Extracellular ATP is an endogenous danger signal that is known to activate inflammatory responses in innate immune cells, including macrophages. Activated macrophages start to secrete proteins to induce an immune response, as well as to recruit other immune cells to the site of infection and tissue damage. In this study, we characterized the secretome (i.e., the global pattern of secreted proteins) of ATP-stimulated human macrophages. We show that ATP stimulation activates robust vesicle-mediated unconventional protein secretion, including exosome release and membrane shedding, from human macrophages. Pathway analysis of the identified secreted proteins showed that calpain-related pathways were overrepresented in the secretome of ATP-stimulated cells. In accordance with this, calpains, which are calcium-dependent nonlysosomal cysteine proteases, were activated upon ATP stimulation through a P2X purinoceptor 7 receptor-dependent pathway. Functional studies demonstrated that calpain activity is essential for the P2X purinoceptor 7 receptor-mediated activation of unconventional protein secretion. Unconventional protein secretion was followed by cell necrosis and NLRP3 inflammasome-mediated secretion of the mature form of the proinflammatory cytokine IL-1β. Furthermore, ATP-driven NLRP3 inflammasome activation was also dependent on calpain activity. Interestingly, pro-IL-1β and inflammasome components ASC and caspase-1 were released by ATP-activated macrophages through a vesicle-mediated secretion pathway. In conclusion, to our knowledge, we provide the first global characterization of proteins secreted by ATP-activated human macrophages and show a pivotal role for calpains in the activation of the inflammatory response during ATP exposure., (Copyright © 2016 by The American Association of Immunologists, Inc.)

Objective: Activation of the inflammasome pathway in macrophages results in the secretion of 2 potent proinflammatory and proatherogenic cytokines, interleukin (IL)-1β, and IL-18. Atherosclerotic lesions are characterized by the presence of various endogenous activators of the NLR family pyrin domain containing 3 (NLRP3) inflammasome, including cholesterol crystals and extracellular ATP. The aim of this study was to comprehensively characterize the expression of inflammasome pathway components and regulators in human atherosclerotic lesions., Approach and Results: Twenty human coronary artery RNA samples from 10 explanted hearts were analyzed using an inflammasome pathway-focused quantitative polymerase chain reaction array. Advanced atherosclerotic plaques, when compared with early-to-intermediate lesions from the same coronary trees, displayed significant upregulation of 12 target genes, including the key inflammasome components apoptosis-associated speck-like protein containing a CARD domain, caspase-1, and IL-18. Immunohistochemical stainings of the advanced plaques revealed macrophage foam cells positive for NLRP3 inflammasome components around the necrotic lipid cores. The polymerase chain reaction array target p38δ mitogen-activated protein kinase was upregulated in advanced plaques and strongly expressed by lesional macrophage foam cells. In cultured human monocyte-derived macrophages, the p38δ mitogen-activated protein kinase was activated by intracellular stress signals triggered during ATP- and cholesterol crystal-induced NLRP3 inflammasome activation and was required for NLRP3-mediated IL-1β secretion., Conclusions: Increased expression of the key inflammasome components in advanced coronary lesions implies enhanced activity of the inflammasome pathway in progression of coronary atherosclerosis. The p38δ mitogen-activated protein kinase was identified as a novel regulator of NLRP3 inflammasome activation in primary human macrophages, and thus, represents a potential target for modulation of atherosclerotic inflammation., (© 2016 American Heart Association, Inc.)

Objective: Apolipoprotein A-I (apoA-I) has been shown to possess several atheroprotective functions, including inhibition of inflammation. Protease-secreting activated mast cells reside in human atherosclerotic lesions. Here we investigated the effects of the neutral proteases released by activated mast cells on the anti-inflammatory properties of apoA-I., Approach and Results: Activation of human mast cells triggered the release of granule-associated proteases chymase, tryptase, cathepsin G, carboxypeptidase A, and granzyme B. Among them, chymase cleaved apoA-I with the greatest efficiency and generated C-terminally truncated apoA-I, which failed to bind with high affinity to human coronary artery endothelial cells. In tumor necrosis factor-α-activated human coronary artery endothelial cells, the chymase-cleaved apoA-I was unable to suppress nuclear factor-κB-dependent upregulation of vascular cell adhesion molecule-1 (VCAM-1) and to block THP-1 cells from adhering to and transmigrating across the human coronary artery endothelial cells. Chymase-cleaved apoA-I also had an impaired ability to downregulate the expression of tumor necrosis factor-α, interleukin-1β, interleukin-6, and interleukin-8 in lipopolysaccharide-activated GM-CSF (granulocyte-macrophage colony-stimulating factor)- and M-CSF (macrophage colony-stimulating factor)-differentiated human macrophage foam cells and to inhibit reactive oxygen species formation in PMA (phorbol 12-myristate 13-acetate)-activated human neutrophils. Importantly, chymase-cleaved apoA-I showed reduced ability to inhibit lipopolysaccharide-induced inflammation in vivo in mice. Treatment with chymase blocked the ability of the apoA-I mimetic peptide L-4F, but not of the protease-resistant D-4F, to inhibit proinflammatory gene expression in activated human coronary artery endothelial cells and macrophage foam cells and to prevent reactive oxygen species formation in activated neutrophils., Conclusions: The findings identify C-terminal cleavage of apoA-I by human mast cell chymase as a novel mechanism leading to loss of its anti-inflammatory functions. When targeting inflamed protease-rich atherosclerotic lesions with apoA-I, infusions of protease-resistant apoA-I might be the appropriate approach., (© 2015 The Authors.)

Objective: In the pathogenesis of coronary atherosclerosis, local macrophage-driven inflammation and secretion of proinflammatory cytokines, interleukin-1β (IL-1β) in particular, are recognized as key factors. Moderate alcohol consumption is associated with a reduced risk of coronary artery disease mortality. Here we examined in cultured human macrophages whether ethanol modulates the intracellular processes involved in the secretion of IL-1β., Results: Ethanol decreased dose-dependently the production of mature IL-1β induced by activators of the NLRP3 inflammasome, i.e. ATP, cholesterol crystals, serum amyloid A and nigericin. Ethanol had no significant effect on the expression of NLRP3 or IL1B mRNA in LPS-primed macrophages. Moreover, secretion of IL-1β was decreased in parallel with reduction of caspase-1 activation, demonstrating that ethanol inhibits inflammasome activation instead of synthesis of pro-IL-1β. Acetaldehyde, a highly reactive metabolite of ethanol, had no effect on the ATP-induced IL-1β secretion. Ethanol also attenuated the secretion of IL-1β triggered by synthetic double-stranded DNA, an activator of the AIM2 inflammasome. Ethanol conferred the inhibitory functions by attenuating the disruption of lysosomal integrity and ensuing leakage of the lysosomal protease cathepsin B and by reducing oligomerization of ASC., Conclusion: Ethanol-induced inhibition of the NLRP3 inflammasome activation in macrophages may represent a biological pathway underlying the protective effect of moderate alcohol consumption on coronary heart disease.

Background: Local acidosis has been demonstrated in ischemic tissues and at inflammatory sites., Results: Acidic extracellular pH triggers NLRP3 inflammasome activation and interleukin-1β secretion in human macrophages., Conclusion: Acidic pH represents a novel danger signal alerting the innate immunity., Significance: Local acidosis may promote inflammation at ischemic and inflammatory sites. Local extracellular acidification has been demonstrated at sites of ischemia and inflammation. IL-1β is one of the key proinflammatory cytokines, and thus, its synthesis and secretion are tightly regulated. The NLRP3 (nucleotide-binding domain leucine-rich repeat containing family, pyrin domain containing 3) inflammasome complex, assembled in response to microbial components or endogenous danger signals, triggers caspase-1-mediated maturation and secretion of IL-1β. In this study, we explored whether acidic environment is sensed by immune cells as an inflammasome-activating danger signal. Human macrophages were exposed to custom cell culture media at pH 7.5-6.0. Acidic medium triggered pH-dependent secretion of IL-1β and activation of caspase-1 via a mechanism involving potassium efflux from the cells. Acidic extracellular pH caused rapid intracellular acidification, and the IL-1β-inducing effect of acidic medium could be mimicked by acidifying the cytosol with bafilomycin A1, a proton pump inhibitor. Knocking down the mRNA expression of NLRP3 receptor abolished IL-1β secretion at acidic pH. Remarkably, alkaline extracellular pH strongly inhibited the IL-1β response to several known NLRP3 activators, demonstrating bipartite regulatory potential of pH on the activity of this inflammasome. The data suggest that acidic environment represents a novel endogenous danger signal alerting the innate immunity. Low pH may thus contribute to inflammation in acidosis-associated pathologies such as atherosclerosis and post-ischemic inflammatory responses.

Aims: Alcohol abuse is associated with increased frequency of infections attributed to ethanol-induced immune suppression. The precise mechanism of immune suppression is however not known. Mast cells (MC) belong to the innate immune system and they have been implicated in the first line of immune defence against bacteria and parasites. Therefore we studied the effects of ethanol and its first metabolite acetaldehyde on mast cell viability, proliferation and apoptosis., Main Methods: Human mast cell line (HMC)-1 cells, mouse bone marrow derived mast cells (mBMMC) and human peripheral blood derived mast cells (HuMC) were used. Effects of ethanol and acetaldehyde on mast cell proliferation were determined by assessing incorporation of [(3)H]thymidine into cellular DNA and by trypan blue exclusion. Apoptosis was assessed by measuring apoptotic nucleosomes and caspase-3, -8 and -9 activities using ELISA and by using Tunel assay. The expression of anti- and proapoptotic proteins Bcl-2 and Bax was analyzed by RT-PCR and western blot, respectively., Key Findings: Ethanol, but not acetaldehyde inhibited dose-dependently the proliferation and viability HMC-1 and mBMMC cells. The decreased viability was caused by apoptotic cell death of the MC. Significant apoptosis of HMC-1 cells was observed in the presence of 43mM (2.5 per thousand) ethanol. Induction of apoptosis was associated with clearly increased caspase-3 activity and moderately increased caspase-8 and 9 activities. Ethanol also shifted the Bcl-2/Bax balance towards apoptosis., Significance: The ethanol-induced reduction of MC viability could contribute to immunosuppression associated with ethanol abuse.

We investigated on-call stress and its consequences among anaesthetists. A questionnaire was sent to all working Finnish anaesthetists (n = 550), with a response rate of 60%. Four categories of on-call workload and a sum variable of stress symptoms were formed. The anaesthetists had the greatest on-call workload among Finnish physicians. In our sample, 68% felt stressed during the study. The most important causes of stress were work and combining work with family. The study showed a positive correlation between stress symptoms and on-call workload (p = 0.009). Moderate burnout was present in 18%vs 45% (p = 0.008) and exhaustion in 32% and 68% (p = 0.015), in the lowest vs highest workload category, respectively. The symptoms were significantly associated with stress, gender, perceived sleep deprivation, suicidal tendencies and sick leave. Being frequently on call correlates with severe stress symptoms and these symptoms are associated with sick leave.

Chemically modified tetracyclines are a group of non-antimicrobial tetracycline derivatives, which possess antiinflammatory, anticollagenolytic and antiproliferative properties. Here we studied the effects of four different chemically modified tetracyclines (CMT-1, CMT-3, CMT-8 and CMT-308) on proliferation and viability of cultured mouse and human mast cells. All studied CMTs (25 microM) effectively inhibited the viability and proliferation of human mast cell line (HMC-1) cells and mouse bone marrow derived mast cells (mBMMCs), as judged by trypan blue exclusion and by incorporation of [(3)H]thymidine. The antiproliferative effect of CMTs was not dependent on the stimulating growth factor, i.e. CMTs inhibited both IL-3 and c-kit ligand-induced proliferation of mBMMCs. The reduced viability of mast cells was due to induction of apoptosis, as indicated by the increased amount of apoptotic nucleosomes and the appearance of TUNEL positive cells in the presence of CMTs. The induction of apoptosis was further confirmed by showing that CMT-3 induces activation of caspase-3 and caspase-9 in HMC-1 cells. Additionally, CMT-3 induced downregulation of the expression of antiapoptotic Bcl-2 protein in HMC-1 cells. Compared to doxycycline, the antiproliferative and proapoptotic effects of different CMTs were clearly more pronounced. Of the studied CMTs, CMT-3 and CMT-8 appeared to be the most potent inhibitors of mast cell proliferation and survival. The present results show that CMTs have an antiproliferative and proapoptotic effect on both malignant and non-malignant mast cells. In conclusion, CMTs could offer a novel means to treat disorders with inappropriate expansion of mast cells, such as rheumatoid arthritis and systemic mast cell diseases.

The flocculation performance of cationic starches on calcite pretreated with anionic sodium polyacrylate (NaPA) was investigated by measuring the mean particle size and the dynamic mobility of the calcite dispersions. Cationic starches of different molecular weight and degree of substitution were used. By varying the amount of anionic sodium polyacrylate, which has a strong affinity to the calcium carbonate surface, one is able to anionically modify the particles and reverse the charge character of the originally cationic calcium carbonate. By such modification of the charge character of the calcium carbonate dispersion, it is possible to approach the mechanisms of flocculation caused by cationic macromolecules like starch. The importance of different mechanisms of flocculation, such as bridging, charge neutralization, and flocculation induced by polyelectrolyte complexes (PEC), was further investigated in this work. It was found that when the NaPA is completely absorbed at the calcite surfaces the mechanism of the flocculation induced by the starch is mainly bridging flocculation. Excess NaPA in the calcium carbonate dispersion will result in polyelectrolyte complexes formed between the non-absorbed NaPA and the oppositely charged starch polymers. These complexes will in most cases strongly enhance the flocculation due to mainly charge neutralization. Depending on the ratio of non-absorbed NaPA and the starch in the aqueous phase, the calcite dispersion is either re-stabilized or more strongly flocculated due to the formed polyelectrolyte complexes. Both the mobility and the particle size measurements support the mechanisms described. It was further demonstrated that the molecular weight and degree of substitution of the starches might be adjusted to control the flocculation behavior.

The distribution pattern of substituents within the granules and the components of two cationised and two oxidised potato starches was studied. The level of crystallinity in wet-cationised (WC) and hypochlorite oxidised (HO) starch granules was similar to that of native starch granules but lower in dry-cationised (DC) and peroxide oxidised (PO) granules. However, the melting temperature of DC granules remained similar to native granules but was decreased in the other samples. With all modified starches, the initial rate of acid hydrolysis (lintnerisation) was increased compared to native granules. The degree of substitution decreased only slightly in WC granules after the lintnerisation, whereas virtually all the substituted glucosyl units in DC starch were hydrolysed already at initial stages. The decrease of substituents in the HO and PO starches was intermediate. The starches were partly resistant to the action of isoamylase and the successive beta-amylolysis, suggesting that substituents were found both close to the branches and near the nonreducing ends in the amylopectin component. It is suggested that the DC starch was preferentially cationised at the surface of the granules, whereas WC and oxidised starches were modified throughout the granules.

The main oxidised component in hypochlorite-oxidised potato starch was isolated by anion-exchange chromatography after enzymatic hydrolysis. The primary structure of the isolated oligosaccharides was determined by 1H and 13C NMR spectroscopy, using homonuclear and heteronuclear two-dimensional techniques. The isolated pentamer and hexamer contained one glucose unit oxidised to a dicarboxyl residue. As the hypochlorite oxidation has occurred at positions C-2 and C-3 of a glucose unit, the introduced carboxyl groups caused ring cleavage between the carbons C-2 and C-3. The ring-cleaved dicarboxyl residue had glycosidic linkages on both sides, implying that this oxidation pathway does not result in depolymerisation. The vicinal coupling constant between H-4 and H-5 in the ring-cleaved dicarboxyl residue was 3.2 Hz, showing that the gauche orientations are preferred. As a result, a different bending of the starch chain is observed and is probably, therefore, one of the reasons why hypochlorite oxidation reduces the tendency to retrogradation. The pKa values (3.0) were determined from the pH-dependent chemical shifts of H-1, H-4 and H-5 of the dicarboxylic residue.

We studied seasonal and between-tree variation in the composition and content of total and individual low-molecular-weight phenolics (LMWP) in leaves of mountain birch trees (Betula pubescens ssp.tortuosa). The major phenolic compounds were chlorogenic acid, quercetin-3-O-β-D-glucuronopyranoside, myricetin-3-O-(5-acetyl)-L-rhamnopyranoside, and 1-O-galloyl-β-D-(2-O-acetyl)-glucopyranose. The content of total phenolics, as well as the sum of individual LMWP, varied only slightly among trees while variation in contents of individual LMWP was large. Concentrations of almost all phenolics decreased during the growing season but pairwise correlations between individual phenolics remained similar over the whole season indicating tree-specific LMWP profiles over the season. Among flavonoids, the between-tree component of variation was 2.6 times as large as the seasonal component, while for variation of nonflavonoids the between-tree component was larger than the seasonal one. To explain the significant correlations within both flavonoid and nonflavonoid compounds, we discuss the biogenesis of LMWP in birch leaves, as well as their ecological role.