naRNA-LL37 composite DAMPs define sterile NETs as self-propagating drivers of inflammation.

Neutrophil extracellular traps (NETs) are a key antimicrobial feature of cellular innate immunity mediated by polymorphonuclear neutrophils (PMNs). NETs counteract microbes but are also linked to inflammation in atherosclerosis, arthritis, or psoriasis by unknown mechanisms. Here, we report that NET-associated RNA (naRNA) stimulates further NET formation in naive PMNs via a unique TLR8-NLRP3 inflammasome-dependent pathway. Keratinocytes respond to naRNA with expression of psoriasis-related genes (e.g., IL17, IL36) via atypical NOD2-RIPK signaling. In vivo, naRNA drives temporary skin inflammation, which is drastically ameliorated by genetic ablation of RNA sensing. Unexpectedly, the naRNA-LL37 'composite damage-associated molecular pattern (DAMP)' is pre-stored in resting neutrophil granules, defining sterile NETs as inflammatory webs that amplify neutrophil activation. However, the activity of the naRNA-LL37 DAMP is transient and hence supposedly self-limiting under physiological conditions. Collectively, upon dysregulated NET release like in psoriasis, naRNA sensing may represent both a potential cause of disease and a new intervention target.

Cracking the NLRP3 code: Pioneering precision medicine for inflammation.

Precisely diagnosing and effectively treating cryopyrin-associated periodic syndrome (CAPS), an inflammatory condition linked to gain-of-function NLRP3 inflammasome mutations, poses challenges. A novel classification approach may help inform therapeutic decisions and offer valuable insights into broader inflammatory conditions (Cosson et al. J. Exp. Med. 2024. https://doi.org/10.1084/jem.20231200).

Inflammasome Activation in Human Macrophages: IL-1ß Cleavage Detection by Fully Automated Capillary-Based Immunoassay.

Interleukin (IL)-1ß is a key mediator of inflammation and activates via pattern recognition receptors (PRR) of the inflammasome family by proteolytic maturation. Proteolysis is driven by proteases such as caspase-1 (also known as IL-1 converting enzyme, ICE) and converts the intact pro-IL-1ß ~31 kDa pro-peptide into a mature, ~17 kDa form that can exit cells through nanomolecular pores or via microvesicles. Whereas pro-IL-1ß fails to trigger IL-1 receptor (IL-1R) activation, mature IL-1ß, upon release from the cell, triggers pleiotropic downstream effects, establishing an inflammatory state. Hence, being able to detect IL-1ß conversion is physiologically relevant for measuring inflammation, but it cannot be easily accomplished by conventional ELISA or flow cytometry as most commercially available antibodies do not discriminate mature and pro-form. Furthermore, unlike for other cytokines, the mere induction and translation of IL1B mRNA cannot serve as a proxy of inflammasome PRR activation. Rather the cleavage of IL-1ß needs to be verified. Hence, conventional immunoblotting has emerged as the gold standard for demonstrating inflammasome activation as the difference in molecular weight between pro- and mature form can easily be detected. However, conventional immunoblotting suffers from poor standardization, quantification, and reproducibility, may require sample concentration, and is also not suitable for medium to high throughput. Some of these shortcomings are prohibitive for analysis of human primary samples but can be overcome by fully automated capillary-based immunoassay as we outline here. We here provide a practical guide to quantify pro- vs mature IL-1ß directly from unconcentrated supernatants of human monocyte-derived macrophages. The assay may be useful for more standardized and medium-throughput analysis in these cells or other biospecimen.

Chitin oligomers promote lymphoid innate and adaptive immune cell activation.

Chitin is a highly abundant N-acetylglucosamine polysaccharide that has been linked to immune responses in the context of fungal infections and allergic asthma, especially to T helper 2 immune responses. Unfortunately, due to the frequent use of crude chitin preparations of unknown purity and degree of polymerization, there is still great uncertainty about how chitin activates different parts of the human immune system. We recently identified chitin oligomers of 6 N-acetylglucosamine units as the smallest immunologically active chitin motif and the innate immune receptor TLR2 as a primary chitin sensor on human and murine myeloid cells, but the response of further immune cells (e.g. lymphoid cells) to oligomeric chitin has not been investigated. Our analysis of primary human immune cells now shows that chitin oligomers activate immune responses of both innate and adaptive lymphocytes: notably, chitin oligomers activated natural killer cells but not B lymphocytes. Moreover, chitin oligomers induced maturation of dendritic cells and enabled potent CD8+ T-cell recall responses. Our results suggest that chitin oligomers not only trigger immediate innate responses in a limited range of myeloid cells but also exert critical activities across the entire human immune system. This highlights chitin oligomer immune activation as an interesting and broadly applicable potential target for both adjuvant development and therapeutic interference in chitin-mediated pathologies.

The role of FIBCD1 in response to Aspergillus fumigatus in lung epithelial cells.

Chitin, a polysaccharide, is ubiquitously found in nature and has been known to be an active immunogen in mammals, and interacts with Toll-like, mannose and glucan receptors, to induce cytokine and chemokine secretions. FIBCD1 is a tetrameric type II transmembrane endocytic vertebrate receptor that binds chitin, is found in human lung epithelium and modulates lung epithelial inflammatory responses to A. fumigatus cell wall polysaccharides. We previously reported the detrimental role of FIBCD1 in a murine model of pulmonary invasive aspergillosis. However, the effect that chitin and chitin-containing A. fumigatus conidia exerts on lung epithelium following exposure through FIBCD1 is not yet fully explored. Using both in vitro and in vivo strategies, we examined how lung and lung epithelial gene expression are modified after exposure to fungal conidia or chitin fragments in the presence or absence of FIBCD1. FIBCD1 expression was associated with a decrease in inflammatory cytokines with increasing size of chitin (dimer-oligomer). Thus, our results demonstrate that FIBCD1 expression modulates cytokine and chemokine expression in response to A. fumigatus conidia that is modified by the presence of chitin particles.

Delayed NLRP3 inflammasome inhibition ameliorates subacute stroke progression in mice.

BACKGROUND: Ischemic stroke immediately evokes a strong neuro-inflammatory response within the vascular compartment, which contributes to primary infarct development under vessel occlusion as well as further infarct growth despite recanalization, referred to as ischemia/reperfusion injury. Later, in the subacute phase of stroke (beyond day 1 after recanalization), further inflammatory processes within the brain parenchyma follow. Whether this second wave of parenchymal inflammation contributes to an additional/secondary increase in infarct volumes and bears the potential to be pharmacologically targeted remains elusive. We addressed the role of the NLR-family pyrin domain-containing protein 3 (NLRP3) inflammasome in the subacute phase of ischemic stroke. METHODS: Focal cerebral ischemia was induced in C57Bl/6 mice by a 30-min transient middle cerebral artery occlusion (tMCAO). Animals were treated with the NLRP3 inhibitor MCC950 therapeutically 24 h after or prophylactically before tMCAO. Stroke outcome, including infarct size and functional deficits as well as the local inflammatory response, was assessed on day 7 after tMCAO. RESULTS: Infarct sizes on day 7 after tMCAO decreased about 35% after delayed and about 60% after prophylactic NLRP3 inhibition compared to vehicle. Functionally, pharmacological inhibition of NLRP3 mitigated the local inflammatory response in the ischemic brain as indicated by reduction of infiltrating immune cells and reactive astrogliosis. CONCLUSIONS: Our results demonstrate that the NLRP3 inflammasome continues to drive neuroinflammation within the subacute stroke phase. NLRP3 inflammasome inhibition leads to a better long-term outcome-even when administered with a delay of 1 day after stroke induction, indicating ongoing inflammation-driven infarct progression. These findings may pave the way for eagerly awaited delayed treatment options in ischemic stroke.

TLR5 Variants Are Associated with the Risk for COPD and NSCLC Development, Better Overall Survival of the NSCLC Patients and Increased Chemosensitivity in the H1299 Cell Line.

Chronic obstructive pulmonary disease (COPD) is considered as the strongest independent risk factor for lung cancer (LC) development, suggesting an overlapping genetic background in both diseases. A common feature of both diseases is aberrant immunity in respiratory epithelia that is mainly regulated by Toll-like receptors (TLRs), key regulators of innate immunity. The function of the flagellin-sensing TLR5 in airway epithelia and pathophysiology of COPD and LC has remained elusive. We performed case−control genetic association and functional studies on the importance of TLR5 in COPD and LC development, comparing Caucasian COPD/LC patients (n = 974) and healthy donors (n = 1283). Association analysis of three single nucleotide polymorphisms (SNPs) (rs725084, rs2072493_N592S, and rs5744174_F616L) indicated the minor allele of rs2072493_N592S to be associated with increased risk for COPD (OR = 4.41, p < 0.0001) and NSCLC (OR = 5.17, p < 0.0001) development and non-small cell LC risk in the presence of COPD (OR = 1.75, p = 0.0031). The presence of minor alleles (rs5744174 and rs725084) in a co-dominant model was associated with overall survival in squamous cell LC patients. Functional analysis indicated that overexpression of the rs2072493_N592S allele affected the activation of NF-κB and AP-1, which could be attributed to impaired phosphorylation of p38 and ERK. Overexpression of TLR5N592S was associated with increased chemosensitivity in the H1299 cell line. Finally, genome-wide transcriptomic analysis on WI-38 and H1299 cells overexpressing TLR5WT or TLR5N592S, respectively, indicated the existence of different transcription profiles affecting several cellular pathways potentially associated with a dysregulated immune response. Our results suggest that TLR5 could be recognized as a potential biomarker for COPD and LC development with functional relevance.

A Baseline Cellular Antiviral State Is Maintained by cGAS and Its Most Frequent Naturally Occurring Variant rs610913.

Upon recognition of aberrantly located DNA, the innate immune sensor cyclic GMP-AMP synthase (cGAS) activates stimulator of IFN genes (STING)/IFN regulatory factor (IRF)3-driven antiviral responses. In this study, we characterized the ability of a specific variant of the human cGAS-encoding gene MB21D1, rs610913, to alter cGAS-mediated DNA sensing and viral infection. rs610913 is a frequent G>T polymorphism resulting in a P261H exchange in the cGAS protein. Data from the International Collaboration for the Genomics of HIV suggested that rs610913 nominally associates with HIV-1 acquisition in vivo. Molecular modeling of cGAS(P261H) hinted toward the possibility for an additional binding site for a potential cellular cofactor in cGAS dimers. However, cGAS(wild-type [WT]) or cGAS(P261H)-reconstituted THP-1 cGAS knockout cells shared steady-state expression of IFN-stimulated genes, as opposed to cells expressing the enzymatically inactive cGAS(G212A/S213A). Accordingly, cGAS(WT) and cGAS(P261H) cells were less susceptible to lentiviral transduction and infection with HIV-1, HSV-1, and Chikungunya virus as compared with cGAS knockout or cGAS(G212A/S213A) cells. Upon DNA challenge, innate immune activation appeared to be mildly reduced upon expression of cGAS(P261H) compared with cGAS(WT). Finally, DNA challenge of PBMCs from donors homozygously expressing rs610913 provoked a trend toward a slightly reduced type I IFN response as compared with PBMCs from GG donors. Taken together, the steady-state activity of cGAS maintains a baseline antiviral state rendering cells more refractory to IFN-stimulated gene-sensitive viral infections. rs610913 failed to grossly differ phenotypically from the WT gene, suggesting that cGAS(P261H) and WT cGAS share a similar ability to sense viral infections in vivo.

Fish TLR5 develops a taste for viral RNA.

These days not only humans but also freshwater fish battle with infections by RNA viruses (Zou & Nie, 2017). This observation prompted Liao et al to turn their attention to viral recognition in Grass carp (Ctenopharyngodon idella), the most important cultivated freshwater fish with 5.7 million tons and 13 billion USD in fishery exports per year (FAO, 2021). Grass carp and other freshwater fish, such as the model organism Danio rerio (zebrafish), have a sophisticated innate immune system that helps them to detect microbial and viral pathogens by employing a variety of pattern recognition receptors (PRRs; Zou & Nie, 2017). Toll-like receptors (TLRs) are one class of PRRs that detect microbe-associated molecular patterns (MAMPs), such as flagellin or viral double-stranded (ds)RNA. In mammals, TLR3 is specialized in sensing viral dsRNA (Liu et al, 2008), while TLR5 recognizes the MAMP flagellin (Yoon et al, 2012; Fig 1). The well-established notion of TLR5 as a purely "bacterial" flagellin TLR has now been challenged by Liao et al in this issue of EMBO Reports (Liao et al, 2022). The authors' intriguing and unexpected results indicate that fish TLR5 is involved in viral recognition, a function lost in mammals, and shed light on hitherto inexplicable links of mammalian TLR5 to antiviral immune signaling.

NLRP3 Phospho-residue Mapping by Phospho Dot Blots.

When characterizing posttranslational modifications like phosphorylation, using efficient screening methods to map the phospho sites is essential, especially when dealing with large multi-domain proteins. NLRP3 (the NOD, LRR, and pyrin domain-containing protein 3), which initiates the formation of an NLRP3 inflammasome complex, is regulated posttranslationally by phosphorylation at several Ser and Tyr residues. However, determining sites of modification are not straightforward. For quick and reliable screening of the candidate phospho sites in NLRP3, we use a phospho dot blot assay which we describe here. This technique employs an in vitro kinase assay with a candidate kinase, Bruton's Tyrosine Kinase (BTK), and peptides derived from the region of interest in the protein that contains the potential phosphorylation sites. The reaction containing the phosphorylated peptides is quickly screened by a dot blot where the peptides are blotted with a commercially available anti-phospho-tyrosine antibody. This method can also be adapted to detect modified Ser or Thr residues and is an ideal screening assay to map phospho residues in NLRP3 or other proteins. This can be an initial screening procedure or can be complemented by other approaches such as site directed mutagenesis and by generating phospho site-specific antibodies.

A Genetic Variation of Lipopolysaccharide Binding Protein Affects the Inflammatory Response and Is Associated with Improved Outcome during Sepsis.

LPS binding protein (LBP) is an important innate sensor of microbial cell wall structures. Frequent functionally relevant mutations exist and have been linked to influence susceptibility to and course of bacterial infections. We examined functional properties of a single nucleotide polymorphism resulting in an exchange of phenylalanine to leucine at position 436 of LBP (rs2232618) and compared the frequent variant of the molecule with the rare one in ligand binding experiments. We then stimulated RAW cells with bacterial ligands in the presence of serum obtained from individuals with different LBP genotypes. We, furthermore, determined the potential effects of structural changes in the molecule by in silico modeling. Finally, we analyzed 363 surgical patients for this genetic variant and examined incidence and course of sepsis following surgery. We found that binding of LBP to bacterial ligands was reduced, and stimulation of RAW cells resulted in an increased release of TNF when adding serum from individuals carrying the F436L variant as compared with normal LBP. In silico analysis revealed structural changes of LBP, potentially explaining some of the effects observed for the LBP variant. Finally, patients carrying the F436L variant were found to be similarly susceptible for sepsis. However, we observed a more favorable course of severe infections in this cohort. Our findings reveal new insights into LPS recognition and the subsequent activation of the innate immune system brought about by LBP. The identification of a genetic variant of LBP influencing the course of sepsis may help to stratify individuals at risk and thus reduce clinical complications of patients.

BTK operates a phospho-tyrosine switch to regulate NLRP3 inflammasome activity.

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-1ß 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.

Absence of Non-Canonical, Inhibitory MYD88 Splice Variants in B Cell Lymphomas Correlates With Sustained NF-κB Signaling.

Gain-of-function mutations of the TLR adaptor and oncoprotein MyD88 drive B cell lymphomagenesis via sustained NF-κB activation. In myeloid cells, both short and sustained TLR activation and NF-κB activation lead to the induction of inhibitory MYD88 splice variants that restrain prolonged NF-κB activation. We therefore sought to investigate whether such a negative feedback loop exists in B cells. Analyzing MYD88 splice variants in normal B cells and different primary B cell malignancies, we observed that MYD88 splice variants in transformed B cells are dominated by the canonical, strongly NF-κB-activating isoform of MYD88 and contain at least three novel, so far uncharacterized signaling-competent splice isoforms. Sustained TLR stimulation in B cells unexpectedly reinforces splicing of NF-κB-promoting, canonical isoforms rather than the 'MyD88s', a negative regulatory isoform reported to be typically induced by TLRs in myeloid cells. This suggests that an essential negative feedback loop restricting TLR signaling in myeloid cells at the level of alternative splicing, is missing in B cells when they undergo proliferation, rendering B cells vulnerable to sustained NF-κB activation and eventual lymphomagenesis. Our results uncover MYD88 alternative splicing as an unappreciated promoter of B cell lymphomagenesis and provide a rationale why oncogenic MYD88 mutations are exclusively found in B cells.

NLRP3 Inflammasome Assembly in Neutrophils Is Supported by PAD4 and Promotes NETosis Under Sterile Conditions.

Neutrophil extracellular trap formation (NETosis) and the NLR family pyrin domain containing 3 (NLRP3) inflammasome assembly are associated with a similar spectrum of human disorders. While NETosis is known to be regulated by peptidylarginine deiminase 4 (PAD4), the role of the NLRP3 inflammasome in NETosis was not addressed. Here, we establish that under sterile conditions the cannonical NLRP3 inflammasome participates in NETosis. We show apoptosis-associated speck-like protein containing a CARD (ASC) speck assembly and caspase-1 cleavage in stimulated mouse neutrophils without LPS priming. PAD4 was needed for optimal NLRP3 inflammasome assembly by regulating NLRP3 and ASC protein levels post-transcriptionally. Genetic ablation of NLRP3 signaling resulted in impaired NET formation, because NLRP3 supported both nuclear envelope and plasma membrane rupture. Pharmacological inhibition of NLRP3 in either mouse or human neutrophils also diminished NETosis. Finally, NLRP3 deficiency resulted in a lower density of NETs in thrombi produced by a stenosis-induced mouse model of deep vein thrombosis. Altogether, our results indicate a PAD4-dependent formation of the NLRP3 inflammasome in neutrophils and implicate NLRP3 in NETosis under noninfectious conditions in vitro and in vivo.

Platelets: Underestimated Regulators of Autoinflammation in Psoriasis.

Platelets have long been known as mediators of hemostasis and, more recently, as mediators of thromboinflammation, although their physiopathological role has mostly been investigated in the context of disease of internal organs, such as liver and kidney, or systemic disorders. Of late, exciting recent data suggest that platelets may also play a role in inflammation at distal sites such as the skin: recent studies show that platelets, by engaging polymorphonuclear neutrophils (PMNs), contribute to local inflammation in the frequent skin disorder, psoriasis. In an experimental model, systemic depletion of platelets drastically attenuated skin inflammation by preventing PMN infiltration of the skin. A broader role of platelets in different types of skin inflammation is therefore likely, and in this paper, we specifically review recent advances in psoriasis. Special emphasis is given to the crosstalk with systemic platelet effects, which may be of interest in psoriasis-related cardiovascular comorbidities. Furthermore, we discuss the potential for platelet-centered interventions in the therapy for psoriasis.

Targeting the NLRP3 Inflammasome via BTK.

The NLRP3 inflammasome represents a critical inflammatory machinery driving pathology in many acute (e. g., myocardial infarction or stroke) and chronic (Alzheimer's disease, atherosclerosis) human disorders linked to the activity of IL-1 cytokines. Although the therapeutic potential of NLRP3 is undisputed, currently no clinically approved therapies exist to target the NLRP3 inflammasome directly. The recent discovery of BTK as a direct and positive regulator of the NLRP3 inflammasome has, however, raised the intriguing possibility of targeting the NLRP3 inflammasome via existing or future BTK inhibitors. Here, I review the mechanistic basis for this notion and discuss the molecular and cellular role of BTK in the inflammasome process. Specific attention will be given to cell-type dependent characteristics and differences that may be relevant for targeting approaches. Furthermore, I review recent (pre-)clinical evidence for effects of BTK inhibitors on NLRP3 activity and highlight and discuss open questions and future research directions. Collectively, the concept of targeting BTK to target NLRP3-dependent inflammation will be explored comprehensively at the molecular, cellular and therapeutic levels.

The NLRP3 inflammasome drives inflammation in ischemia/reperfusion injury after transient middle cerebral artery occlusion in mice.

PURPOSE: Cerebral ischemia induces a profound neuro-inflammatory response, but the underlying molecular mechanisms are poorly understood. Inflammasomes (NLRP1, NLRP3, NLRC4, AIM2) are intracellular multi-protein complexes which can induce sets of pro-inflammatory cyto- and chemokines, and thereby guide inflammation. We, here, assessed the functional role of NLRP3 in ischemia/reperfusion (I/R) injury in a mouse model of transient cerebral ischemia. METHODS: Ischemic stroke was induced in C57Bl/6 mice by 60 min transient middle cerebral artery occlusion (tMCAO) and 3, 7 or 23 h of reperfusion, a paradigm of I/R injury. The expression patterns of inflammasomes in the ischemic hemispheres were evaluated by semiquantitative real-time PCR and Western Blot analysis accompanied by protein localization using immunocytochemistry. Finally, animals were treated with the inflammasome inhibitors Sulforaphane, Genipin, MCC950 or vehicle, directly before or upon recanalization after tMCAO. Stroke outcome was assessed, including infarct size and functional deficits, local inflammatory response, neuronal survival as well as blood-brain barrier function on day 1 after tMCAO. RESULTS: After tMCAO the relative gene expression levels of NLRP3 increased 20-30x within 1 day in the ischemic hemisphere which translated into an increased expression of NLRP3 in neurons. Accordingly, the gene expression levels of the NLRP3-modulator, Bruton's Tyrosine Kinase (BTK), and the NLRP3-inducible cytokine IL-1ß significantly rose. Lesser or non-significant changes were seen for the other inflammasomes. Application of inflammasome inhibitors covering all inflammasomes or specifically NLRP3 significantly reduced infarct volumes when given before or after tMCAO and was accompanied by clear evidence for reduced activation of caspase 1. This stroke attenuating effect coincided with less immune cell infiltration in the ischemic hemisphere and preservation of the blood-brain barrier integrity. CONCLUSIONS: Our data show that induction of the NLRP3 inflammasome in neurons drives neuroinflammation in acute ischemic stroke. Early blockade of NLRP3 protects from I/R injury by mitigating inflammation and stabilizing the blood-brain barrier.

Recent insights into the regulatory networks of NLRP3 inflammasome activation.

The NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome is a fascinating cellular machinery endowed with the capacity for rapid proteolytic processing of the pro-inflammatory cytokine IL-1ß and the cell death effector gasdermin D (GSDMD). Although its activity is essential to fight infection and support tissue homeostasis, the inflammasome complex, which consists of the danger sensor NLRP3, the adaptor apoptosis-associated speck-like protein containing a CARD (ASC; also known as PYCARD), caspase-1 and probably other regulatory proteins, also bears considerable potential for detrimental inflammation, as observed in human conditions such as gout, heart attack, stroke and Alzheimer's disease. Thus, multi-layered regulatory networks are required to ensure the fine balance between rapid responsiveness versus erroneous activation (sufficient and temporally restricted versus excessive and chronic activity) of the inflammasome. These involve multiple activation, secretion and cell death pathways, as well as modulation of the subcellular localization of NLRP3, and its structure and activity, owing to post-translational modification by other cellular proteins. Here, we discuss the exciting progress that has recently been made in deciphering the regulation of the NLRP3 inflammasome. Additionally, we highlight open questions and describe areas of research that warrant further exploration to obtain a more comprehensive molecular and cellular understanding of the NLRP3 inflammasome.