Pulmonary vascular inflammation with fatal coronavirus disease 2019 (COVID-19): possible role for the NLRP3 inflammasome.

BACKGROUND: Pulmonary hyperinflammation is a key event with SARS-CoV-2 infection. Acute respiratory distress syndrome (ARDS) that often accompanies COVID-19 appears to have worse outcomes than ARDS from other causes. To date, numerous lung histological studies in cases of COVID-19 have shown extensive inflammation and injury, but the extent to which these are a COVID-19 specific, or are an ARDS and/or mechanical ventilation (MV) related phenomenon is not clear. Furthermore, while lung hyperinflammation with ARDS (COVID-19 or from other causes) has been well studied, there is scarce documentation of vascular inflammation in COVID-19 lungs. METHODS: Lung sections from 8 COVID-19 affected and 11 non-COVID-19 subjects, of which 8 were acute respiratory disease syndrome (ARDS) affected (non-COVID-19 ARDS) and 3 were from subjects with non-respiratory diseases (non-COVID-19 non-ARDS) were H&E stained to ascertain histopathological features. Inflammation along the vessel wall was also monitored by expression of NLRP3 and caspase 1. RESULTS: In lungs from COVID-19 affected subjects, vascular changes in the form of microthrombi in small vessels, arterial thrombosis, and organization were extensive as compared to lungs from non-COVID-19 (i.e., non-COVID-19 ARDS and non-COVID-19 non-ARDS) affected subjects. The expression of NLRP3 pathway components was higher in lungs from COVID-19 ARDS subjects as compared to non-COVID-19 non-ARDS cases. No differences were observed between COVID-19 ARDS and non-COVID-19 ARDS lungs. CONCLUSION: Vascular changes as well as NLRP3 inflammasome pathway activation were not different between COVID-19 and non-COVID-19 ARDS suggesting that these responses are not a COVID-19 specific phenomenon and are possibly more related to respiratory distress and associated strategies (such as MV) for treatment.

Oral intake of silica nanoparticles exacerbates intestinal inflammation.

Nanoparticles, i.e., particles with a diameter of ≤100 nm regardless of their composing material, are added to various foods as moisturizers, coloring agents, and preservatives. Silicon dioxide (SiO2, silica) nanoparticles in particular are widely used as food additives. However, the influence of SiO2 nanoparticle oral consumption on intestinal homeostasis remains unclear. The daily intake of 10-nm-sized SiO2 nanoparticles exacerbates dextran sulfate sodium (DSS)-induced colitis, whereas the daily intake of 30-nm-sized SiO2 nanoparticles has no influence on intestinal inflammation. The exacerbation of colitis induced by consuming 10-nm-sized SiO2 nanoparticles was abolished in mice deficient in apoptosis-associated speck-like protein containing a CARD (ASC). Our study indicates that the oral intake of small SiO2 nanoparticles poses a risk for worsening intestinal inflammation through activation of the ASC inflammasome.

Discovery of carbon-11 labeled sulfonamide derivative: A PET tracer for imaging brain NLRP3 inflammasome.

We report herein the discovery of a positron emission tomography (PET) tracer for the (NOD)-like receptor protein 3 (NLRP3). Our recent medicinal chemistry campaign on developing sulfonamide-based NLRP3 inhibitors led to an analog, 1, with a methoxy substituent amenable to labeling with carbon-11. PET/CT imaging studies indicated that [11C]1 exhibited rapid blood-brain barrier (BBB) penetration and moderate brain uptake, as well as blockable uptake in the brain. [11C]1, thus suggesting the potential to serve as a useful tool for imaging NLRP3 inflammasome in living brains.

NLRP3 inflammasome negatively regulates podocyte autophagy in diabetic nephropathy.

Inflammasome mechanisms are recognized as a key pathophysiology of diabetic nephropathy (DN). The nucleotide-oligomerization domain-like receptor 3 (NLRP3) inflammasome has attracted the most attention. Autophagy as a conserved intracellular catabolic pathway plays essential roles in the maintenance of podocytes. Although autophagy was involved in preventing excessive inflammatory responses in kidney diseases, a clear understanding of the regulation of NLRP3 inflammasome on autophagy in glomerular damage in DN is still lacking. In this study, we focused on the effect of the activation of NLRP3 inflammasome on the suppression of podocyte autophagy and aimed to investigate the role of autophagy in podocyte injury in DN. Podocyte autophagy has been confirmed to be inhibited in high-fat diet/streptozotocin (HFD/STZ)-induced DN mice, and NLRP3 has been found to be upregulated in both mice and human DN biopsies and in vitro. Activation of NLRP3 inflammasome exacerbated podocyte autophagy and reduced podocyte nephrin expression, while silencing of NLRP3 efficiently restored podocyte autophagy and ameliorated podocyte injury induced by high glucose. The results showed that NLRP3 was a negative regulator of autophagy and suggested that restoration of podocyte autophagy by inactivation of NLRP3 under high glucose could reduce podocyte injury. Proper modification of autophagy and inflammasome has the potential to benefit the kidney in DN.

Estrogen receptors participate in carcinogenesis signaling pathways by directly regulating NOD-like receptors.

Increasing evidence indicates that the NOD-like receptors (NLRs) family may act as critical back-up defenses and provide synergistic responses when confronted with persistent danger. However, the precise regulatory mechanism of NLRs and the contribution of NLRs to cancer are still unknown. In our previous study, we found that estrogen receptors (ERs) have a close connection with NLRs in the inflammatory response. Here, ERs are first identified as NLRs transcription regulation factors, both regulate NLRs expression and promote inflammasome co-localization. Furthermore, we identified that NLRP3 was differentially expressed in colon normal and cancer cells, selective ERα antagonist could significantly decrease pro-inflammatory cytokines expression, suppress proliferation and promote apoptosis by inhibited NLRP3 expression and inflammasome activity. In short, the research demonstrates that ERs participate in the NLR-associated signaling pathway in cancer by directly regulating NLRs. Our results provide novel insight into ERs as therapeutic targets in NLR-related inflammation and cancer.

A longitudinal study highlights shared aspects of the transcriptomic response to cardiogenic and septic shock.

BACKGROUND: Septic shock (SS) and cardiogenic shock (CS) are two types of circulatory shock with a different etiology. Several studies have described the molecular alterations in SS patients, whereas the molecular factors involved in CS have been poorly investigated. We aimed to assess in the whole blood of CS and SS patients, using septic patients without shock (SC) as controls, transcriptomic modifications that occur over 1 week after ICU admission and are common to the two types of shock. METHODS: We performed whole blood RNA sequencing in 21 SS, 11 CS, and 5 SC. In shock patients, blood samples were collected within 16 h from ICU admission (T1), 48 h after ICU admission (T2), and at day 7 or before discharge (T3). In controls, blood samples were available at T1 and T2. Gene expression changes over time have been studied in CS, SS, and SC separately with a paired analysis. Genes with p value < 0.01 (Benjamini-Hochberg multiple test correction) were defined differentially expressed (DEGs). We used gene set enrichment analysis (GSEA) to identify the biological processes and transcriptional regulators significantly enriched in both types of shock. RESULTS: In both CS and SS patients, GO terms of inflammatory response and pattern recognition receptors (PRRs) were downregulated following ICU admission, whereas gene sets of DNA replication were upregulated. At the gene level, we observed that alarmins, interleukin receptors, PRRs, inflammasome, and DNA replication genes significantly changed their expression in CS and SS, but not in SC. Analysis of transcription factor targets showed in both CS and SS patients, an enrichment of CCAAT-enhancer-binding protein beta (CEBPB) targets in genes downregulated over time and an enrichment of E2F targets in genes with an increasing expression trend. CONCLUSIONS: This pilot study supports, within the limits of a small sample size, the role of alarmins, PRRs, DNA replication, and immunoglobulins in the pathophysiology of circulatory shock, either in the presence of infection or not. We hypothesize that these genes could be potential targets of therapeutic interventions in CS and SS. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02141607. Registered 19 May 2014.

The selective Nlrp3 inflammasome inhibitor Mcc950 attenuates lung ischemia-reperfusion injury.

Lung ischemia-reperfusion (IR) occurs in many circumstances and leads to impaired lung function. The NACHT, LRR and PYD domains-containing protein 3 (Nlrp3) inflammasome is reportedly activated during lung IR. Mcc950 is a recently developed Nlrp3 inhibitor. The aim of our study was to test the efficacy of Mcc950 on lung IR injury and to investigate the role of reactive oxygen species (ROS) in Nlrp3 inflammasome activation using a murine lung IR model. The results of the current study confirmed that Nlrp3 was upregulated and activated during lung IR, and inhibiting oxidative stress by the ROS scavenger edaravone attenuated Nlrp3 inflammasome activation. Mcc950 pretreatment significantly alleviated IR-induced lung injury by reducing production of the proinflammatory cytokines Il-1ß and Il-18 and inhibiting neutrophil infiltration and cell apoptosis. Protein coimmunoprecipitation revealed that Mcc950 partially blocked the interaction between Nlrp3 and Nek7 (NimA-related protein kinase 7). Therefore, we conclude that ROS-dependent activation of the Nlrp3 inflammasome contributed to lung IR injury. Mcc950 significantly reduced lung IR injury by blocking Nlrp3 inflammasome activation, and the mechanism was partially attributed to inhibition of the interaction between Nlrp3 and Nek7. Thus, Mcc950 is a promising treatment for the prevention of lung IR injury.

A novel flow cytometric method to assess inflammasome formation.

Inflammasomes are large protein complexes induced by a wide range of microbial, stress, and environmental stimuli that function to induce cell death and inflammatory cytokine processing. Formation of an inflammasome involves dramatic relocalization of the inflammasome adapter protein apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) into a single speck. We have developed a flow cytometric assay for inflammasome formation, time of flight inflammasome evaluation, which detects the change in ASC distribution within the cell. The transit of ASC into the speck is detected by a decreased width or increased height of the pulse of emitted fluorescence. This assay can be used to quantify native inflammasome formation in subsets of mixed cell populations ex vivo. It can also provide a rapid and sensitive technique for investigating molecular interactions in inflammasome formation, by comparison of wild-type and mutant proteins in inflammasome reconstitution experiments.

Roles of the NLRP3 inflammasome in the pathogenesis of diabetic nephropathy.

Diabetic nephropathy (DN) is a serious complication of diabetes mellitus, and persistent inflammation in circulatory and renal tissues is an important pathophysiological basis for DN. The essence of the microinflammatory state is the innate immune response, which is central to the occurrence and development of DN. Members of the inflammasome family, including both "receptors" and "regulators", are key to the inflammatory immune response. Nucleotide binding and oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) and other inflammasome components are able to detect endogenous danger signals, resulting in activation of caspase-1 as well as interleukin (IL)-1ß, IL-18 and other cytokines; these events stimulate the inflammatory cascade reaction, which is crucial for DN. Hyperglycaemia, hyperlipidaemia and hyperuricaemia can activate the NLRP3 inflammasome, which then mediates the occurrence and development of DN through the K+ channel model, the lysosomal damage model and the active oxygen cluster model. In this review, we survey the involvement of the NLRP3 inflammasome in various signalling pathways and highlight different aspects of their influence on DN. We also explore the important effects of the NLRP3 inflammasome on kidney function and structural changes that occur during DN development and progression. It is becoming more evident that NLRP3 inflammasome targeting has therapeutic potential for the treatment of DN.

Activation of the NLRP3/caspase-1 inflammasome in human dental pulp tissue and human dental pulp fibroblasts.

The NLRP3/caspase-1 inflammasome pathway plays an important role in cellular immune defence against bacterial infection; however, its function in human dental pulp tissue and human dental pulp fibroblasts remains poorly understood. We demonstrate that NLRP3 protein expression occurs to a greater extent in pulp tissue with irreversible pulpitis than in normal pulp tissue and in tissue with reversible pulpitis. Caspase-1 is present in its active (cleaved) form only in pulp tissue with irreversible pulpitis. NLRP3 and caspase-1 are expressed in the odontoblast layers in normal human dental pulp tissue, whereas in inflamed pulp tissue, the odontoblast layers are disrupted and dental pulp cells are positive for NLRP3 and caspase-1. Additionally, we investigate the role of the NLRP3/caspase-1 inflammasome pathway in human dental pulp fibroblasts and show that ATP activates the P2X7 receptor on the cell membrane triggering K(+) efflux and inducing the gradual recruitment of the membrane pore pannexin-1. Extracellular lipopolysaccharide is able to penetrate the cytosol and activate NLRP3. Furthermore, the low intracellular K(+) concentration in the cytosol triggers reactive oxygen species generation, which also induces the NLRP3 inflammasome. Thus, the NLRP3/caspase-1 pathway has a biological role in the innate immune response mounted by human dental pulp fibroblasts.

Innate immunity but not NLRP3 inflammasome activation correlates with severity of stable COPD.

BACKGROUND: In models of COPD, environmental stressors induce innate immune responses, inflammasome activation and inflammation. However, the interaction between these responses and their role in driving pulmonary inflammation in stable COPD is unknown. OBJECTIVES: To investigate the activation of innate immunity and inflammasome pathways in the bronchial mucosa and bronchoalveolar lavage (BAL) of patients with stable COPD of different severity and control healthy smokers and non-smokers. METHODS: Innate immune mediators (interleukin (IL)-6, IL-7, IL-10, IL-27, IL-37, thymic stromal lymphopoietin (TSLP), interferon γ and their receptors, STAT1 and pSTAT1) and inflammasome components (NLRP3, NALP7, caspase 1, IL-1ß and its receptors, IL-18, IL-33, ST2) were measured in the bronchial mucosa using immunohistochemistry. IL-6, soluble IL-6R, sgp130, IL-7, IL-27, HMGB1, IL-33, IL-37 and soluble ST2 were measured in BAL using ELISA. RESULTS: In bronchial biopsies IL-27+ and pSTAT1+ cells are increased in patients with severe COPD compared with control healthy smokers. IL-7+ cells are increased in patients with COPD and control smokers compared with control non-smokers. In severe stable COPD IL-7R+, IL-27R+ and TSLPR+ cells are increased in comparison with both control groups. The NALP3 inflammasome is not activated in patients with stable COPD compared with control subjects. The inflammasome inhibitory molecules NALP7 and IL-37 are increased in patients with COPD compared with control smokers. IL-6 levels are increased in BAL from patients with stable COPD compared with control smokers with normal lung function whereas IL-1ß and IL-18 were similar across all groups. CONCLUSIONS: Increased expression of IL-27, IL-37 and NALP7 in the bronchial mucosa may be involved in progression of stable COPD.

NOD-like receptors in intestinal homeostasis and epithelial tissue repair.

The intestinal epithelium constitutes a dynamic physical barrier segregating the luminal content from the underlying mucosal tissue. Following injury, the epithelial integrity is restored by rapid migration of intestinal epithelial cells (IECs) across the denuded area in a process known as wound healing. Hence, through a sequence of events involving restitution, proliferation and differentiation of IECs the gap is resealed and homeostasis reestablished. Relapsing damage followed by healing of the inflamed mucosa is a hallmark of several intestinal disorders including inflammatory bowel diseases (IBD). While several regulatory peptides, growth factors and cytokines stimulate restitution of the epithelial layer after injury, recent evidence in the field underscores the contribution of innate immunity in controlling this process. In particular, nucleotide-binding and oligomerization domain-like receptors (NLRs) play critical roles in sensing the commensal microbiota, maintaining homeostasis, and regulating intestinal inflammation. Here, we review the process of intestinal epithelial tissue repair and we specifically focus on the impact of NLR-mediated signaling mechanisms involved in governing epithelial wound healing during disease.

Protocol for measuring NLRC4 inflammasome activation and pyroptosis in murine bone-marrow-derived macrophages.

NLR family CARD domain containing protein 4 (NLRC4) inflammasome activation and the associated pyroptosis are critical for protection against infection by bacterial pathogens. This protocol presents a detailed procedure to activate and measure NLRC4 inflammasome activation and pyroptosis upon Salmonella Typhimurium infection. The techniques can be adapted to monitoring the activation of other types of inflammasomes and pathogenic stimuli. For comprehensive details on the use and execution of this protocol, please refer to Dong et al. (2021).

Measuring the Inflammasome in Oncogene-Induced Senescence.

Inflammasomes are multimeric protein complexes that process IL-1ß by cleaving the translated full-length protein into its active IL-1ß mature fragment. In oncogene-induced senescence, inflammasomes play a crucial role by regulating IL1R signaling and consequently modulating proliferation and the senescence-associated secretory phenotype (SASP). Inflammasome activation requires two steps: (a) priming of the inflammasome by activation of IL1B expression, followed by (b) cleavage and release of mature IL-1ß. In this chapter, we describe methods to detect both stages of inflammasome activation in cellular senescence.

Analysis of Inflammasome Activation in Response to Yersinia Infection by Fluorescence Microscopy Detection of Active Caspase-1 Puncta.

The type of cell death triggered by a particular environmental stimulus influences the outcome of infection or inflammatory disease processes. The ability to identify the cell death pathway that is activated in response to infection is essential for understanding the pathogenesis and host response to infection. Activation of the cysteine protease caspase-1 in various inflammasome complexes indicates that cells are undergoing pyroptosis, a regulated, proinflammatory cell death. Inflammasome assembly and caspase activation can be measured by various methods ranging from detection of inflammasome-dependent cell death, cytokine secretion, cleavage of caspase-1, or the formation of "puncta" within the cell that contain inflammasome components, such as caspase-1 or the adapter protein ASC. Here we describe a method for detecting caspase-1 activation on a single cell level in the context of infection by the Gram-negative pathogen Yersinia using immunofluorescence microscopy. We previously used this approach to quantify caspase-1 puncta formation in cells containing Yersinia translocon components (Zwack et al., MBio 6:e02095-14, 2015). This is a modification of methods used previously by Broz et al. (Cell Host Microbe 8:471-483, 2010) and Case and Roy (MBio 2:e00117-11, 2011). By taking a microscopy-based approach that allows us to quantify puncta as well as other cell-biological features of infection (i.e., number of bacteria associated with a particular cell; levels of bacterial effector or translocon proteins in caspase-1 puncta-containing cells; or levels or localization of host cellular proteins), we can better quantify the heterogeneity between cells undergoing pyroptosis and cells that are not under the same infection conditions. These approaches have the potential to generate hypotheses that can enable further mechanistic insight into activation of pyroptosis in response to bacterial infection.

Methods for Detection of Pyrin Inflammasome Assembly in Macrophages Infected with Yersinia spp.

The Yersinia effector proteins YopE and YopT are important bacterial virulence factors that are secreted into infected host cells and can inactivate Rho GTPases, like RhoA, Rac1, and Cdc42. In order to compensate for the consequences of this effect, the host cell can sense RhoA modifications and trigger a proinflammatory reaction to control the infection. This host response, known as pyrin inflammasome assembly, is normally prevented by another important effector, YopM, allowing Yersinia to counteract this conserved innate immune response. Once assembled, the pyrin inflammasome can activate caspase-1 via proteolysis, leading to IL-1ß secretion and cell death through pyroptosis. Here we describe how to measure pyrin inflammasome assembly, in response to YopE or YopT activities, when macrophages are infected with yopM mutant Yersinia. Using primary mouse macrophages as host cells, we show how to detect this host response through the downstream events of pyrin dephosphorylation, caspase-1 proteolysis, IL-1ß release, and pyroptosis.

Role of the thioredoxin interacting protein in diabetic nephropathy and the mechanism of regulating NOD­like receptor protein 3 inflammatory corpuscle.

Inflammatory response serves an important role in diabetic nephropathy (DN); however, the mechanism of inflammatory response results in renal damage is not yet clear. The aim of the present study was to investigate the role of the thioredoxin interacting protein (TXNIP)/NOD­like receptor protein 3 (NLRP3) axis­mediated activation of NLRP3 inflammasome in DN. A diabetic rat model was induced by streptozotocin injection. Hematoxylin and eosin (H&E) staining and streptavidin­peroxidase staining were then used to examine the kidney tissue morphology, and TXNIP and NLRP3 protein expression levels, respectively. Furthermore, RNA interference technology was applied to silence the TXNIP gene. TXNIP and NLRP3 inflammasome activation­associated proteins and mRNAs were detected by western blot analysis and RT­qPCR, respectively. Enzyme­linked immunosorbent assay was also used to examine interleukin (IL)­1 and IL­18 expression, while flow cytometry was performed to detect reactive oxygen species production. The data revealed that TXNIP and NLRP3 were overexpressed in kidney tissue of DN rats, and the level of antioxidant genes was downregulated. It was also observed that glucose promoted TXNIP expression and activation of NLRP3 inflammasome in a time­dependent and dose­dependent manner, therefore promoting inflammatory responses. Silencing of TXNIP gene resulted in the downregulation of NLRP3 inflammasome activation, and inhibited the expression levels of IL­1 and IL­18 in a high­glucose environment. Furthermore, low expression of TXNIP promoted the levels of antioxidant genes and reduced the ROS levels. Taken together, the high­glucose environment was able to upregulated the level of inflammatory factors by promoting the expression of TXNIP and the activation of NLRP3 inflammasome, consequently participating in DN.

Measuring Innate Immune Responses to Bacterial Viability.

The innate immune system directly senses microbial viability via the detection of a special class of viability-associated pathogen-associated molecular patterns (vita-PAMPs), such as prokaryotic messenger RNA. In the case of Gram-negative bacteria, detection of bacterial viability by phagocytes leads to a unique activation of inflammasome and type I interferon pathways, resulting in a robust pro-inflammatory innate response and a vigorous adaptive immune response. This protocol describes the methods required to study activation of both inflammasome and type I interferon pathways after stimulation of mouse bone marrow-derived macrophages with live or killed Gram-negative and Gram-positive bacteria. It covers the generation and handling of bone marrow-derived macrophages, the culture and killing of bacteria, the preparation of bacterial messenger RNA, and the stimulation of macrophages with live or killed bacteria. Lastly, this protocol describes the techniques employed to measure the hallmarks of inflammasome (secretion of interleukin-1ß) and type I interferon (activation of TBK1, IRF3 and secretion of type I interferon) pathways.

Detection of ASC Speck Formation by Flow Cytometry and Chemical Cross-linking.

Assembly of a relatively large protein aggregate or "speck" formed by the adaptor protein ASC is a common downstream step in the activation of most inflammasomes. This unique feature of ASC allows its visualization by several imaging techniques and constitutes a reliable and feasible readout for inflammasome activation in cells and tissues. We have previously described step-by-step protocols to generate immortalized cell lines stably expressing ASC fused to a fluorescent protein for measuring inflammasome activation by confocal microscopy, and immunofluorescence of endogenous ASC in primary cells. Here, we present two more methods to detect ASC speck formation: (1) Assessment of ASC speck formation by flow cytometry; and (2) Chemical cross-linking of ASC followed by immunoblotting. These methods allow for the discrimination of inflammasome-activated versus non-activated cells, the identification of lineage-specific inflammasome activation in complex cell mixtures, and sorting of inflammasome-activated cells for further analysis.

NLRP3 inflammasome signaling as an early molecular response is negatively controlled by miR-186 in CFA-induced prosopalgia mice.

The NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome is the most frequently studied in the central nervous system and has been linked to neuropathic pain. In this study, a post-translational mechanism of microRNA (miR)-186 via regulating the expression of NLRP3 in the complete Freund's adjuvant (CFA)-treated mice was investigated. The injection of CFA was used to induce trigeminal neuropathic pain in mice. miRs microarray chip assay was performed in trigeminal ganglions (TGs). CFA treatment significantly increased the mRNA expression of NLRP3, interleukin (IL)-1ß, and IL-18 in TGs compared to the control group. Moreover, 26 miRs were differentially expressed in TGs from trigeminal neuropathic pain mice, and the expression of miR-186 showed the lowest level of all the miRs. Further examination revealed that NLRP3 was a candidate target gene of miR-186. We delivered miR-186 mimics to CFA-treated mice. The head withdrawal thresholds of the CFA-treated mice were significantly increased by miR-186 mimics injection compared with CFA single treatment. The mRNA and protein expression of NLRP3, IL-1ß, and IL-18 in TGs from trigeminal neuropathic pain mice were significantly inhibited by miR-186 mimics treatment compared to the CFA group. miR-186 was able to suppress the neuropathic pain via regulating the NLRP3 inflammasome signaling.