Inflammasome formation in the lungs of patients with fatal COVID-19.

OBJECTIVE: The orf8b protein of the coronavirus SARS-CoV, analogous to SARS-CoV-2, triggers the NLRP3 inflammasome in macrophages in vitro. Deregulated inflammasome-mediated release of interleukin-1 family cytokines is important in hyper-inflammatory syndromes, like happens in SARS-CoV-2-mediated cytokine release syndrome. We propose that an intense inflammasome formation characterizes the lungs of patients with fatal COVID-19 disease due to pneumonia and acute respiratory distress syndrome (ARDS). METHODS: Samples from four patients with confirmed COVID-19 pneumonia who had been hospitalized at the Hospital of the University of Trieste (Italy) and died of ARDS and four lung samples from a historical repository from subjects who had died of cardiopulmonary arrest and had not been placed on mechanical ventilation and without evidence of pulmonary infection at postmortem examination were collected. Pathology samples had been fixed in formalin 10% at time of collection and subsequently embedded in paraffin. We conducted staining for ASC (Apoptosis-associated Speck-like protein containing a Caspase recruitment domain), NLRP3 (NACHT, LRR, and PYD domains-containing protein 3), and cleaved caspase-1. RESULTS: Intense expression of the inflammasome was detected, mainly in leukocytes, within the lungs of all patients with fatal COVID-19 in the areas of lung injury. The number of ASC inflammasome specks per high power fields was significantly higher in the lungs of patients with fatal COVID-19 as compared with the lungs of control subjects (52 ± 22 vs 6 ± 3, P = 0.0064). CONCLUSIONS: These findings identify the presence of NLRP3 inflammasome aggregates in the lungs of fatal COVID-19 pneumonia thus providing the potential molecular link between viral infection and cytokine release syndrome.

Effects of hyperbaric oxygen on NLRP3 inflammasome activation in the brain after carbon monoxide poisoning.

Neuroinflammation plays an important role in brain damage after acute carbon monoxide poisoning (ACOP). The nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing (NLRP) 3 inflammasome triggers the activation of inflammatory caspases and maturation of interleukin (IL)-1ß and -18, and has been linked to various human autoinflammatory and autoimmune diseases. In this study we investigated the effects of hyperbaric oxygen (HBO2) on NLRP3 inflammasome activation after ACOP. Mice were randomly divided into four groups: sham group (exposure to normobaric air - i.e., 21% O2 at 1 atmosphere absolute); HBO2-only group; CO + normobaric air group; and CO + HBO2 group. Cognitive function was evaluated with the Morris water maze; myelin injury was assessed by FluoroMyelin GreenTM fluorescent myelin staining and myelin basic protein (MBP) immunostaining; and mRNA and protein levels of NLRP3 inflammasome complex proteins were measured by quantitative real-time PCR and Western blot, respectively. Additionally, serum and brain levels of IL-1ßß and -18 and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase were determined by enzyme-linked immunosorbent assay. It was found that HBO2 improved learning and memory, and alleviated myelin injury in mice subjected to acute CO exposure. Furthermore, HBO2 decreased NLRP3, absent in melanoma 2 (AIM2), caspase-1, and apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain mRNA and protein levels, and reduced brain and serum concentrations of IL-1ß and -18 and NADPH oxidase. These results indicate that HBO2 suppresses the inflammatory response after ACOP by blocking NLRP3 inflammasome activation, thereby alleviating cognitive deficits.

Upregulation of the NLRC4 inflammasome contributes to poor prognosis in glioma patients.

Inflammation in tumor microenvironments is implicated in the pathogenesis of tumor development. In particular, inflammasomes, which modulate innate immune functions, are linked to tumor growth and anticancer responses. However, the role of the NLRC4 inflammasome in gliomas remains unclear. Here, we investigated whether the upregulation of the NLRC4 inflammasome is associated with the clinical prognosis of gliomas. We analyzed the protein expression and localization of NLRC4 in glioma tissues from 11 patients by immunohistochemistry. We examined the interaction between the expression of NLRC4 and clinical prognosis via a Kaplan-Meier survival analysis. The level of NLRC4 protein was increased in brain tissues, specifically, in astrocytes, from glioma patients. NLRC4 expression was associated with a poor prognosis in glioma patients, and the upregulation of NLRC4 in astrocytomas was associated with poor survival. Furthermore, hierarchical clustering of data from the Cancer Genome Atlas dataset showed that NLRC4 was highly expressed in gliomas relative to that in a normal healthy group. Our results suggest that the upregulation of the NLRC4 inflammasome contributes to a poor prognosis for gliomas and presents a potential therapeutic target and diagnostic marker.

Inflammasome and Caspase-1 Activity Characterization and Evaluation: An Imaging Flow Cytometer-Based Detection and Assessment of Inflammasome Specks and Caspase-1 Activation.

Inflammasome dysregulation is a hallmark of various inflammatory diseases. Evaluating inflammasome-associated structures (ASC specks) and caspase-1 activity by microscopy is time consuming and limited by small sample size. The current flow cytometric method, time of flight inflammasome evaluation (TOFIE), cannot visualize ASC specks or caspase-1 activity, making colocalization studies of inflammasome components and enzymatic activity impossible. We describe a rapid, high-throughput, single-cell, fluorescence-based image analysis method utilizing the Amnis ImageStreamX instrument that quantitatively and qualitatively characterizes the frequency, area, and cellular distribution of ASC specks and caspase-1 activity in mouse and human cells. Unlike TOFIE, this method differentiates between singular perinuclear specks and false positives. With our technique we also show that the presence of NLRP3 reduces the size of ASC specks, which is further reduced by the presence of active caspase-1. The capacity of our approach to simultaneously detect and quantify ASC specks and caspase-1 activity, both at the population and single-cell level, renders it the most powerful tool available for visualizing and quantifying the impact of mutations on inflammasome assembly and activity.

Quantification of Inflammasome Adaptor Protein ASC in Biological Samples by Multiple-Reaction Monitoring Mass Spectrometry.

Inflammation is an integral component of many diseases, including chronic kidney disease (CKD). ASC (apoptosis-associated speck-like protein containing CARD, also PYCARD) is the key inflammasome adaptor protein in the innate immune response. Since ASC specks, a macromolecular condensate of ASC protein, can be released by inflammasome-activated cells into the extracellular space to amplify inflammatory responses, the ASC protein could be an important biomarker in diagnostic applications. Herein, we describe the development and validation of a multiple reaction monitoring mass spectrometry (MRM-MS) assay for the accurate quantification of ASC in human biospecimens. Limits of detection and quantification for the signature DLLLQALR peptide (used as surrogate for the target ASC protein) were determined by the method of standard addition using synthetic isotope-labeled internal standard (SIS) peptide and urine matrix from a healthy donor (LOQ was 8.25 pM, with a ~ 1000-fold linear range). We further quantified ASC in the urine of CKD patients (8.4 ± 1.3 ng ASC/ml urine, n = 13). ASC was positively correlated with proteinuria and urinary IL-18 in CKD samples but not with urinary creatinine. Unfortunately, the ASC protein is susceptible to degradation, and patient urine that was thawed and refrozen lost 85% of the ASC signal. In summary, the MRM-MS assay provides a robust means to quantify ASC in biological samples, including clinical biospecimens; however, sample collection and storage conditions will have a critical impact on assay reliability.

Evidence of inflammasome activation and formation of monocyte-derived ASC specks in HIV-1 positive patients.

OBJECTIVE: The formation of large intracellular protein aggregates of the inflammasome adaptor protein ASC (apoptosis-associated speck-like protein containing a caspase-recruitment domain; also know as PYCARD) is a hallmark of inflammasome activation. ASC speck-forming cells release the highly proinflammatory cytokine IL-1ß in addition to ASC specks into the extracellular space during pyroptotic cell death. There ASC specks can propagate inflammation to other nonactivated cells or tissues. HIV-1 retroviral infection triggers inflammasome activation of abortively infected CD4⁺ T cells in secondary lymphatic tissues. However, if pyroptosis occurs in other peripheral blood mononuclear cells (PBMCs) of HIV-1-infected patients is currently unknown. We investigated if ASC speck positive cells are present in the circulation of HIV-1-infected patients. DESIGN AND METHODS: PBMCs or plasma of HIV-1 infected, antiretroviral therapy-naive patients were analyzed for the presence of ASC speck⁺ cells or extracellular ASC and compared with healthy controls. Intracellular staining for ASC was employed to detect ASC speck⁺ cells within PBMCs by flow cytometry, and ELISA to detect free ASC in the plasma. ASC multimerization was confirmed by immunoblot. RESULTS: Peripheral blood CD14⁺⁺CD16⁻ monocytes were ASC speck⁺ in HIV patients, but not in healthy controls. In the subgroup analysis, HIV patients with lower CD4⁺ T-cell counts and higher viral load had significantly more ASC speck⁺ monocytes. ASC speck formation did not correlate with Gag expression, coinfection, lactate dehydrogenase or C-reactive protein. CONCLUSION: Our findings suggest that pyroptotic CD14⁺⁺CD16⁻ classical monocytes of HIV-1-infected patients release ASC specks into the blood stream, a phenomenon that may contribute to HIV-1 induced inflammation and immune activation.

[RETRACTED ARTICLE] Therapeutic effect and potential mechanism of pioglitazone in rats with severe acute pancreatitis

Caspase recruitment domain-containing protein 9 (Card9) is located upstream of the nuclear factor kappa B (NF-κB) and p38 mitogen-activated protein kinase (MAPK) inflammatory pathways. This study investigated the therapeutic effect and potential mechanism of pioglitazone in rats with severe acute pancreatitis (SAP). SAP was induced by a retrograde infusion of 5.0% sodium taurocholate into the biliopancreatic duct of Sprague Dawley rats (n=54), which were then treated with pioglitazone. Blood and pancreatic tissues were harvested at 3, 6, and 12 h after SAP induction. Pancreatic pathological damage was evaluated by hematoxylin and eosin staining. Serum amylase, serum pro-inflammatory cytokines, and pancreatic myeloperoxidase (MPO) activities were determined by enzyme-linked immunosorbent assay. The expression of Card9 mRNA and protein in pancreatic tissues was detected by real-time polymerase chain reaction and western blotting. Pioglitazone had a therapeutic effect in treating rats with SAP by decreasing the level of amylase activity, ameliorating pancreatic histological damage, decreasing serum pro-inflammatory cytokine levels and tissue MPO activity, and downregulating the expression of NF-κB, p38MAPK, and Card9 mRNAs and proteins (P<0.05). The present study demonstrated that the inhibition of Card9 expression could reduce the severity of SAP. Card9 has a role in the pathogenic mechanism of SAP.

Therapeutic effect and potential mechanism of pioglitazone in rats with severe acute pancreatitis.

Caspase recruitment domain-containing protein 9 (Card9) is located upstream of the nuclear factor kappa B (NF-κB) and p38 mitogen-activated protein kinase (MAPK) inflammatory pathways. This study investigated the therapeutic effect and potential mechanism of pioglitazone in rats with severe acute pancreatitis (SAP). SAP was induced by a retrograde infusion of 5.0% sodium taurocholate into the biliopancreatic duct of Sprague Dawley rats (n=54), which were then treated with pioglitazone. Blood and pancreatic tissues were harvested at 3, 6, and 12 h after SAP induction. Pancreatic pathological damage was evaluated by hematoxylin and eosin staining. Serum amylase, serum pro-inflammatory cytokines, and pancreatic myeloperoxidase (MPO) activities were determined by enzyme-linked immunosorbent assay. The expression of Card9 mRNA and protein in pancreatic tissues was detected by real-time polymerase chain reaction and western blotting. Pioglitazone had a therapeutic effect in treating rats with SAP by decreasing the level of amylase activity, ameliorating pancreatic histological damage, decreasing serum pro-inflammatory cytokine levels and tissue MPO activity, and downregulating the expression of NF-κB, p38MAPK, and Card9 mRNAs and proteins (P<0.05). The present study demonstrated that the inhibition of Card9 expression could reduce the severity of SAP. Card9 has a role in the pathogenic mechanism of SAP.

Salivary Levels of NLRP3 Inflammasome-Related Proteins as Potential Biomarkers of Periodontal Clinical Status.

BACKGROUND: Emerging evidence suggests that activation of inflammasomes plays a central mechanism in pathogenesis of periodontitis. This study aims to compare salivary levels of nod-like receptor family pyrin domain containing protein (NLRP) 3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), cysteine aspartase (caspase)-1, and interleukin (IL)-1ß from individuals with aggressive (AgP) or chronic periodontitis (CP) and healthy controls (HC), as well as elucidate its association with periodontal clinical status. METHODS: Saliva samples from individuals with CP (n = 75), AgP (n = 20), and HC (n = 69) were collected. Periodontal status was assessed by measurement of probing depth, clinical attachment level, and extent and severity of disease. Salivary levels of analytes were analyzed by enzyme-linked immunosorbent assay. Association between biomarkers with CP or AgP was analyzed using multivariate binary logistic regression models. RESULTS: Significantly higher levels of NLRP3, ASC, and IL-1ß were detected in periodontitis groups in comparison to the periodontally HC group. However, no significant differences were observed for caspase-1 levels between clinical groups, and only NLRP3 salivary concentration was significantly higher in AgP compared with CP patients. Also, positive significant correlations among NLRP3, ASC, and IL-1ß salivary concentrations and clinical parameters were observed. Logistic regression analyses revealed a strong/independent association of NLRP3, ASC, and IL-1ß salivary levels with CP and AgP. CONCLUSION: Although the concentration of caspase-1 in saliva samples makes its determination useless for detection of periodontal disease and/or its severity, salivary levels of NLRP3, ASC, and IL-1ß may act as strong/independent indicators of amount and extent of periodontal breakdown in both CP and AgP and could potentially be used for prevention and therapy of this group of diseases.

[Protective effect of taxifolin on H2O2-induced H9C2 cell pyroptosis].

OBJECTIVE: To explore the effect of taxifolin on H2O2-induced pyroptosis in H9C2 cells and the possible mechanisms.
 Methods: The H9C2 cells was divided into 3 groups: a control group, a hydrogen peroxide (H2O2)group and a taxifolin group. The morphology of H9C2 cells was observed by inverted phase contrast microscope. The mitochondrial membrane potential was measured by JC-1 staining and flow cytometry. The alteration of the level of reactive oxygen species (ROS) was detected by specific mitochondrial probe. The protein levels of cysteinyl aspartate specific proteinase-1 (caspase-1)was determined by Western blot. The mRNA levels of interleukin-18 (IL-18), interleukin-1a (IL-1a), interleukin-1b (IL-1b), absent in melanoma 2 (AIM2), apoptosis-associated apeck-like protein (ASC), nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3)and nucleotide-binding oligomerization domain-like receptor family caspase recruitment domain-containing protein 4 (NLRC4) were determined by reverse transcription-polymerase chain reaction (RT-PCR).
 Results: Compared with the control group, the morphology of H9C2 cells obviously changed in the H2O2-treated group, which was guadually improved in the presence of taxifolin. Compared with the control group, the mitochondrial membrane potential was markedly decreased in the H2O2-treated cells, accompanied by the increase ofROS (both P<0.05). Compared with the H2O2 group, the mitochondrial membrane potential changes in the taxifolin group was increased while the ROS was decreased, with significant difference (both P<0.05). Compared with the control group, the protein level of caspase-1 and the mRNA levels of IL-18, IL-1a, IL-1b, AIM2, ASC, NLRP3 and NLRC4 in the H2O2-treated group were significantly increased (all P<0.05), which were attenuated in the presence of taxifolin (all P<0.05).
 Conclusion: Taxifolin can protect H9C2 cells from oxidative injury, and it is able to suppress the H2O2-induced H9C2 cell pyroptosis through inhibition of AIM2, NLRP3 and NLRC4 in flammasome.

Ellagic Acid-Changed Epigenome of Ribosomal Genes and Condensed RPA194-Positive Regions of Nucleoli in Tumour Cells.

We studied the effect of ellagic acid (EA) on the morphology of nucleoli and on the pattern of major proteins of the nucleolus. After EA treatment of HeLa cells, we observed condensation of nucleoli as documented by the pattern of argyrophilic nucleolar organizer regions (AgNORs). EA also induced condensation of RPA194-positive nucleolar regions, but no morphological changes were observed in nucleolar compartments positive for UBF1/2 proteins or fibrillarin. Studied morphological changes induced by EA were compared with the morphology of control, non-treated cells and with pronounced condensation of all nucleolar domains caused by actinomycin D (ACT-D) treatment. Similarly as ACT-D, but in a lesser extent, EA induced an increased number of 53BP1-positive DNA lesions. However, the main marker of DNA lesions, γH2AX, was not accumulated in body-like nuclear structures. An increased level of γH2AX was found by immunofluorescence and Western blots only after EA treatment. Intriguingly, the levels of fibrillarin, UBF1/2 and γH2AX were increased at the promoters of ribosomal genes, while 53BP1 and CARM1 levels were decreased by EA treatment at these genomic regions. In the entire genome, EA reduced H3R17 dimethylation. Taken together, ellagic acid is capable of significantly changing the nucleolar morphology and protein levels inside the nucleolus.

Bringing statistics up to speed with data in analysis of lymphocyte motility.

Two-photon (2P) microscopy provides immunologists with 3D video of the movement of lymphocytes in vivo. Motility parameters extracted from these videos allow detailed analysis of lymphocyte motility in lymph nodes and peripheral tissues. However, standard parametric statistical analyses such as the Student's t-test are often used incorrectly, and fail to take into account confounds introduced by the experimental methods, potentially leading to erroneous conclusions about T cell motility. Here, we compare the motility of WT T cell versus PKCθ-/-, CARMA1-/-, CCR7-/-, and PTX-treated T cells. We show that the fluorescent dyes used to label T cells have significant effects on T cell motility, and we demonstrate the use of factorial ANOVA as a statistical tool that can control for these effects. In addition, researchers often choose between the use of "cell-based" parameters by averaging multiple steps of a single cell over time (e.g. cell mean speed), or "step-based" parameters, in which all steps of a cell population (e.g. instantaneous speed) are grouped without regard for the cell track. Using mixed model ANOVA, we show that we can maintain cell-based analyses without losing the statistical power of step-based data. We find that as we use additional levels of statistical control, we can more accurately estimate the speed of T cells as they move in lymph nodes as well as measure the impact of individual signaling molecules on T cell motility. As there is increasing interest in using computational modeling to understand T cell behavior in in vivo, these quantitative measures not only give us a better determination of actual T cell movement, they may prove crucial for models to generate accurate predictions about T cell behavior.

Overexpression of CARMA3 is associated with advanced tumor stage, cell cycle progression, and cisplatin resistance in human epithelial ovarian cancer.

CARD recruited membrane associated protein 3 (CARMA3) overexpression has been found in several human cancers. However, its expression pattern and biological roles in human ovarian cancers are not clear. In this study, we examined the expression pattern of CARMA3 in 101 ovarian cancer specimens. We found that 52 (51.5 %) showed CARMA3 overexpression. CARMA3 overexpression positively correlated with tumor histology and advanced FIGO stage. CARMA3 depletion in ovarian cancer cell lines A2780 and HO8910 inhibited ovarian cancer cell proliferation and blocked cell cycle progression. CARMA3 depletion also sensitized ovarian cancer cells to cisplatin-induced cytotoxicity. In addition, Western blot showed that CARMA3 depletion downregulated cyclin D1, cyclin E, and Bcl-2 levels. In conclusion, our data provides evidence that CARMA3 is overexpressed in ovarian cancers and associated with advanced stage. CARMA3 regulates the ovarian cancer cell proliferation, cell cycle progression, and chemoresistance.

CARMA3 is upregulated in human pancreatic carcinoma, and its depletion inhibits tumor proliferation, migration, and invasion.

Elevated CARMA3 expression has been reported to be involved in tumor progression of several cancer types. In the present study, we examined the expression pattern of CARMA3 protein and its biological roles in human pancreatic carcinoma. Using immunohistochemistry, we checked CARMA3 protein expression in 95 pancreatic ductal carcinoma specimens. We found that CARMA3 was overexpressed in 34 of 95 (35.8 %) specimens. A significant association was observed between CARMA3 overexpression with histological grade (p=0.0099) and nodal status (p=0.0126). To further explore its biological roles, we knocked down CARMA3 expression in CAPAN2 cell line using small interfering RNA (siRNA). MTT growth assay, wound healing assay, and Transwell assay showed that CARMA3 depletion inhibited cell proliferation, migration, and invasion. We also showed that CARMA3 depletion inhibited EGF-induced nuclear factor-kappaB (NF-κB) activation and its target genes' expression. The effect of CARMA3 depletion on NF-κB signaling was significantly reduced in Bcl10-depleted cells. In conclusion, CARMA3 is overexpressed in pancreatic cancer and regulates malignant cell growth, invasion, and NF-κB signaling, which was dependent on its association with Bcl10.

Clinical significance and biological roles of CARMA3 in human bladder carcinoma.

Caspase recruitment domain and membrane-associated guanylate kinase-like domain protein 3 (CARMA3) was reported as an oncoprotein overexpressed in several cancers. The expression pattern of CARMA3 and its clinical significance in human bladder cancer have not been well characterized. In the present study, CARMA3 expression was analyzed in 90 archived bladder cancer specimens using immunohistochemistry, and the correlation between CARMA3 expression and clinicopathological parameters was evaluated. We found that CARMA3 was overexpressed in 35 of 90 (38.8%) bladder cancer specimens. Significant association was observed between CARMA3 overexpression with tumor status (p = 0.081) and tumor grade (p = 0.027). To further explore the biological functions of CARMA3 in bladder cancer, we depleted CARMA3 in T24 and 5637 cell lines using small interfering RNA (siRNA). Using cell counting kit-8 (CCK8) assay and colony formation assay, we were able to show that CARMA3 depletion inhibited cell proliferation and colony number. Further study demonstrated that CARMA3 depletion decreased an expression of nuclear factor kappa B (NF-κB) targets cyclin D1 and Bcl-2 expression, as well as IκB phosphorylation. Luciferase reporter assay showed that CARMA3 depletion could downregulate NF-κB reporter activity. In conclusion, CARMA3 is overexpressed in bladder cancer and regulates malignant cell growth and NF-κB signaling, which makes CARMA3 a candidate therapeutic target for bladder cancer.