Unraveling the blood microbiome: novel insights into inflammasome responses in Crohn's disease.

OBJECTIVE: Crohn's disease (CD), an inflammatory bowel disease with unknown etiology, is influenced by genetic, environmental, and immunological factors. This study aimed to analyze the blood microbiome and inflammasome responses, emphasizing NLRP3 protein expression and IL-1ß and IL-18 plasma levels, between Crohn's patients and healthy subjects. METHODS: A total of 40 volunteers were included in this study. The 16S rRNA technique was used to sequence the V3-V4 regions of the blood sample. NLRP3 protein levels in plasma were ascertained through Western Blot, and IL-1ß and IL-18 plasma profiles were examined using ELISA. RESULTS: Analysis highlighted five unique phyla in patients' plasma, emphasizing the role of the blood microbiome in CD. Compared to controls, Crohn's patients exhibited elevated NLRP3 protein expression. Plasma IL-1ß levels were diminished in patients ( P  = 0.0041), whereas IL-18 levels were comparably higher ( P  = 0.8209). In patients with CD, the presence of Staphylococcus sciuri in blood samples highlights its potential role in the disease's onset. The study also underscored the interplay between dietary habits, specifically increased meat consumption, and the progression of CD. CONCLUSION: Our pioneering research discerns the variations in the blood microbiome and inflammasome responses between Crohn's patients and healthy individuals. Significant microbiome alterations and the detection of the Staphylococcus sciuri pathogen in Crohn's patients were notable. The pronounced NLRP3 protein in patients suggests its potential as a diagnostic biomarker. Future explorations into IL-1ß and IL-18 pathways promise to unveil innovative insights into CD.

Activation of the AIM2 Receptor in Circulating Cells of Post-COVID-19 Patients With Signs of Lung Fibrosis Is Associated With the Release of IL-1α, IFN-α and TGF-ß.

Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), responsible for COVID-19, has caused a global pandemic. Observational studies revealed a condition, herein called as Long-COVID syndrome (PC), that affects both moderately and severely infected patients, reducing quality-of-life. The mechanism/s underlying the onset of fibrotic-like changes in PC are still not well defined. The goal of this study was to understand the involvement of the Absent in melanoma-2 (AIM2) inflammasome in PC-associated lung fibrosis-like changes revealed by chest CT scans. Peripheral blood mononuclear cells (PBMCs) obtained from PC patients who did not develop signs of lung fibrosis were not responsive to AIM2 activation by Poly dA:dT. In sharp contrast, PBMCs from PC patients with signs of lung fibrosis were highly responsive to AIM2 activation, which induced the release of IL-1α, IFN-α and TGF-ß. The recognition of Poly dA:dT was not due to the activation of cyclic GMP-AMP (cGAMP) synthase, a stimulator of interferon response (cGAS-STING) pathways, implying a role for AIM2 in PC conditions. The release of IFN-α was caspase-1- and caspase-4-dependent when AIM2 was triggered. Instead, the release of pro-inflammatory IL-1α and pro-fibrogenic TGF-ß were inflammasome independent because the inhibition of caspase-1 and caspase-4 did not alter the levels of the two cytokines. Moreover, the responsiveness of AIM2 correlated with higher expression of the receptor in circulating CD14+ cells in PBMCs from patients with signs of lung fibrosis.

Characterizing the NLRP3 Inflammasome in Mood Disorders: Overview, Technical Development, and Measures of Peripheral Activation in Adolescent Patients.

The NOD-, LRR-, and pyrin-domain-containing protein 3 (NLRP3) inflammasome is a node of intracellular stress pathways and a druggable target which integrates mitochondrial stress and inflammatory cascades. While a body of evidence suggests the involvement of the NLRP3 inflammasome in numerous diseases, a lack of reliable measurement techniques highlights the need for a robust assay using small quantities of biological samples. We present a literature overview on peripheral activation of the NLRP3 inflammasome in mood disorders, then outline a process to develop and validate a robust assay to measure baseline and activated intracellular levels of "apoptosis-associated speck-like protein containing a CARD" (ASC) as a key component of an inflammatory profile in peripheral blood mononuclear cells (PBMC). A consistent association between high NLRP3 mRNA levels and relevant cytokines was seen in the literature. Using our method to measure ASC, stimulation of PBMC with lipopolysaccharide and nigericin or adenosine triphosphate resulted in microscopic identification of intracellular ASC specks, as well as interleukin 1 (IL-1) beta and caspase-1 p10 in the periphery. This was abolished by dose-dependent pre-treatment with 100 nM MCC950. We also report the use of this technique in a small pilot sample from patients with bipolar disorder and depressive disorders. The results show that levels of intracellular ASC and IL-1 beta are sensitive to change upon activation and maintained over time, which may be used to improve the detection of NLRP3 activation and guide personalized therapeutic strategy in the treatment of patients.

Endotoxin Acts Synergistically With Clostridioides difficile Toxin B to Increase Interleukin 1ß Production: A Potential Role for the Intestinal Biome in Modifying the Severity of C. difficile Colitis.

BACKGROUND: Inflammation is a crucial driver of host damage in patients with Clostridioides difficile colitis. We examined the potential for the intestinal microbiome to modify inflammation in patients with C. difficile colitis via the effects of gut-derived endotoxin on cytokine production. METHODS: Endotoxin from Escherichia coli and Pseudomonas aeruginosa as well as stool-derived endotoxin were tested for their ability to enhance interleukin 1ß (IL-1ß) and tumor necrosis factor alpha (TNF-α) production by toxin B-stimulated peripheral blood mononuclear cells. Inflammasome and Toll-like receptor 4 (TLR4) blocking studies were done to discern the importance of these pathways, while metagenomic studies were done to characterize predominant organisms from stool samples. RESULTS: Endotoxin significantly enhanced the ability of C. difficile toxin B to promote IL-1ß production but not TNF-α. The magnitude of this effect varied by endotoxin type and was dependent on combined inflammasome and TLR4 activation. Stool-derived endotoxin exhibited a similar synergistic effect on IL-1ß production with less synergy observed for stools that contained a high proportion of γ-proteobacteria. CONCLUSIONS: The ability of endotoxin to enhance IL-1ß production highlights a manner by which the microbiome can modify inflammation and severity of C. difficile disease. This information may be useful in devising new therapies for severe C. difficile colitis.

Research Note: Phytobiotics modulate the expression profile of circulating inflammasome and cyto(chemo)kine in whole blood of broilers exposed to cyclic heat stress.

Heat stress (HS) is a critical concern to the poultry industry as it affects both productivity and well-being. Various managerial and nutritional strategies have been proposed to mitigate the negative effects of HS in chickens, with plant-based additives showing promise. Recently, we reported the positive effect of a phytogenic feed additive (PFA) on growth performance in HS birds. Owing to the antioxidant nature of these compounds, we sought to further explore the effect of PFA on whole blood circulating chemokines, cytokines, and inflammasomes in HS broilers. Broilers (600 males, 1 d) were randomly assigned to 12 environmental chambers, subjected to 2 environmental conditions (12 h cyclic heat stress, HS, 35°C vs. thermoneutral condition [TN], 24°C) and fed 3 diets (control, PFA-C 250 ppm, PFA-C 400 ppm) in a 2 × 3 factorial design. After 21 d of cyclic HS, blood samples were collected for target gene expression analysis. HS upregulated the expression of superoxide dismutase 1 (SOD1) and downregulated glutathione peroxidase-3 (GPX-3), and there was diet × temperature interaction for SOD2, GPX-1, and GPX-3, where gene expression was increased by PFA-C250 during HS but was unchanged for PFA-C400. Plasma total antioxidant capacity (TAC) and malondialdehyde (MDA) content were increased by HS. Gene expression of interleukin-18 (IL-18) was decreased by HS, without further effect of PFA. HS increased tumor necrosis factor α (TNFα), but this effect was mitigated by PFA-C400. C-C motif chemokine ligands 4 and 20 (CCL4 and CCL20) showed a similar pattern to TNFα, with PFA-C400 ameliorating the negative effect of HS. The nucleotide-binding, leucine-rich repeat and pyrin domain containing 3 (NLRP3) inflammasome was decreased by HS and further lowered by PFA-C400, but the nucleotide-binding oligomerization domain, leucine-rich repeat, and CARD domain containing 3 (NLRC3) and nucleotide-binding, leucine-rich repeat containing X1 (NLRX1) inflammasomes were increased by PFA under TN conditions, with no effects of HS. Heat shock proteins (HSP) and heat shock factors (HSF) were unaffected by PFA or HS. Together these data indicate that gene expression of circulating inflammatory factors are dysregulated during HS, and supplemental dietary PFA may be protective.

Quartz Dust Exposure Affects NLRP3 Inflammasome Activation and Plasma Levels of IL-18 and IL-1Ra in Iron Foundry Workers.

PURPOSE: To study the association between inhalation of particulate matter or quartz in Swedish iron foundries and the effects on NLRP3 inflammasome activation. METHODS: Particle exposure measurements were performed during an eight-hour work day for 85 foundry workers at three Swedish iron foundries. Personal sampling was used for measurement of respirable quartz and dust and stationary measurements to obtain exposure measurements for inhalable dust and PM10. The NLRP3 inflammasome markers, interleukin- (IL-) 1ß and IL-18, and inhibitors IL-1 receptor antagonist (IL-1Ra) and IL-18 binding protein (IL-18BP) were measured in plasma. Inflammasome activation was measured by caspase-1 enzymatic activity in monocytes in whole blood by flow cytometry, and expression of inflammasome-related genes was quantified using real-time PCR. Multiple linear regression analysis was used to investigate associations between PM exposures and inflammatory markers. Sex, age, smoking, current infection, BMI, and single nucleotide polymorphism in the inflammasome regulating genes CARD8 (C10X) and NLRP3 (Q705K) were included as covariates. RESULTS: The average exposure levels of respirable dust and quartz were 0.85 and 0.052 mg/m3, respectively. A significant exposure-response was found for respirable dust and IL-18 and for inhalable dust and IL-1Ra. Whole blood, drawn from study participants, was stimulated ex vivo with inflammasome priming stimuli LPS or Pam3CSK4, resulting in a 47% and 49% increase in caspase-1 enzymatic activity in monocytes. This increase in caspase-1 activity was significantly attenuated in the higher exposure groups for most PM exposure measures. CONCLUSIONS: The results indicate that exposure levels of PM in the iron foundry environment can affect the NLRP3 inflammasome and systemic inflammation.

NLRP3 inflammasome and related cytokines reflect the immune status of patients with HBV-ACLF.

BACKGROUND: The NLRP3 inflammasome has been suggested to play a crucial role in host antiviral defense, including against hepatitis B virus (HBV) infection. In the present study, we measured expression of NLRP3 and its related cytokines in patients with different stages of HBV-related acute-on-chronic liver failure (HBV-ACLF), a pattern of end-stage liver disease that occurs frequently in patients with chronic HBV (CHB) infection or HBV-related cirrhosis. METHODS: A total of 75 subjects including 30 HBV-ACLF patients, 30 CHB patients, and 15 healthy controls (HCs) were enrolled. The NLRP3 inflammasome and its components (caspase-1, interleukin (IL)-1ß, and IL-18) were measured in peripheral blood mononuclear cells (PBMCs), macrophages, and liver using flow cytometry, quantitative real-time polymerase chain reaction (RT-PCR), western blot, and immunohistochemistry. The LPS was used to evaluate changes in NLRP3 and its related cytokines in CD14+ monocytes which may reflect immune status. Cytokine expression was measured using RT-PCR. RESULTS: Patients with HBV-ACLF had lower NLRP3 inflammasome expression in peripheral CD14+ monocytes, particularly in the middle-to-late stage, but higher expression in liver macrophages compared to CHB and HCs. Compared with H-LPS or L-LPS alone, L-LPS sequential H-LPS can significantly inhibit the expression of NLRP3 and its related cytokines. CONCLUSION: Differential expression patterns of the NLRP3 inflammasome in the periphery and liver might be related to immune dysfunction and recruitment of monocytes to the injured liver during disease progression. Persistent systemic inflammation is likely a cause of compromised immune status in patients with HBV-ACLF.

Sera from Septic Patients Contain the Inhibiting Activity of the Extracellular ATP-Dependent Inflammasome Pathway.

Immunoparalysis is a common cause of death for critical care patients with sepsis, during which comprehensive suppression of innate and adaptive immunity plays a significant pathophysiological role. Although the underlying mechanisms are unknown, damage-associated molecular patterns (DAMPs) from septic tissues might be involved. Therefore, we surveyed sera from septic patients for factors that suppress the innate immune response to DAMPs, including adenosine triphosphate (ATP), monosodium urate, and high mobility group box-1. Macrophages, derived from THP-1 human acute monocytic leukemia cells, were incubated with each DAMP, in the presence or absence of sera that were collected from critically ill patients. Secreted cytokines were then quantified, and cell lysates were assayed for relevant intracellular signaling mediators. Sera from septic patients who ultimately did not survive significantly suppressed IL-1ß production only in response to extracellular ATP. This effect was most pronounced with sera collected on day 3, and persisted with sera collected on day 7. However, this effect was not observed when THP-1 cells were treated with sera from survivors of sepsis. Septic sera collected at the time of admission (day 1) also diminished intracellular levels of inositol 1,4,5-triphosphate and cytosolic calcium (P < 0.01), both of which are essential for ATP signaling. Finally, activated caspase-1 was significantly diminished in cells exposed to sera collected on day 7 (P < 0.05). In conclusion, the sera of septic patients contain certain factors that persistently suppress the immune response to extracellular ATP, thereby leading to adverse clinical outcomes.

Pentoxifylline inhibits TLR- and inflammasome-mediated in vitro inflammatory cytokine production in human blood with greater efficacy and potency in newborns.

BACKGROUND: Toll-like receptor (TLR)-mediated inflammation may contribute to neonatal sepsis, for which pentoxifylline (PTX), a phosphodiesterase inhibitor that raises intracellular cAMP, is a candidate adjunctive therapy. We characterized the anti-inflammatory effects of PTX toward TLR-mediated production of inflammatory (tumor necrosis factor (TNF) and interleukin (IL)-1ß) and proresolution (IL-6 and IL-10) cytokines in human newborn and adult blood. METHODS: Newborn cord and adult blood were treated with PTX (50-400 µmol/l) before, during or after stimulation with LPS (TLR4 agonist), R848 (TLR7/8 agonist) or LPS/ATP (inflammasome activation). Cytokines were measured by multiplex assay (supernatants), intracellular cytokines and signaling molecules by flow cytometry, and mRNA by quantitative real-time PCR. RESULTS: Whether added 2 h pre-, simultaneously to, or 2 h post-TLR stimulation, PTX inhibited TLR-mediated cytokine production in a concentration-dependent manner, with greater efficacy and potency in newborn blood, decreasing intracellular TNF and IL-1ß with relative preservation of IL-10 and IL-6. PTX decreased TLR-mediated TNF mRNA while increasing IL-10 mRNA. Neonatal plasma factors contributed to the anti-inflammatory effects of PTX in newborn blood that were independent of soluble TNF receptor concentrations, p38 MAPK phosphorylation and IĸB degradation. CONCLUSION: PTX is a potent and efficacious inhibitor of TLR-mediated inflammatory cytokines in newborn cord blood and a promising neonatal anti-inflammatory agent.

Cofactor-independent antiphospholipid antibodies activate the NLRP3-inflammasome via endosomal NADPH-oxidase: implications for the antiphospholipid syndrome.

The antiphospholipid syndrome (APS) is an autoimmune disease characterised by thromboembolic events and/or pregnancy morbidity in the presence of antiphospholipid antibodies (aPL). Here we show that three cofactor independent human monoclonal aPL can induce transcription of NLRP3 and caspase-1 resulting in inflammasome activation specific for NLRP3. This depends fully on activation of endosomal NADPH-oxidase-2 (NOX2) by aPL. Activation of NOX2 and subsequent inflammasome activation by aPL are independent from TLR2 or TLR4. While endosomal superoxide production induces caspase-1 and NLRP3 transcription, it does not affect prae-IL-1ß transcription. Therefore, release of IL-1ß occurs only after activation of additional pathways like TLR7/8 or TLR2. All effects exerted by the monoclonal aPL can be reproduced with IgG fractions of APS patients proving that the monoclonal aPL are representative for the APS. IgG fractions of healthy controls or patients suffering from systemic lupus erythematosus have no effect. In a mouse model of the APS we can show inflammasome activation in vivo. Furthermore, mononuclear cells isolated from patients with the APS show an increased expression of caspase-1 and NLRP3 which is accompanied by a three-fold increased serum concentration of IL-1ß suggesting chronic inflammasome activation in APS patients. In summary, we provide further evidence that endosomal NOX2 can be activated by cofactor independent aPL. This leads to induction of the NLRP3 inflammasome. Our data indicate that cofactor independent aPL might contribute significantly to the pathogenesis of the APS.

The NLRP3 inflammasome is released as a particulate danger signal that amplifies the inflammatory response.

Assembly of the NLRP3 inflammasome activates caspase-1 and mediates the processing and release of the leaderless cytokine IL-1ß and thereby serves a central role in the inflammatory response and in diverse human diseases. Here we found that upon activation of caspase-1, oligomeric NLRP3 inflammasome particles were released from macrophages. Recombinant oligomeric protein particles composed of the adaptor ASC or the p.D303N mutant form of NLRP3 associated with cryopyrin-associated periodic syndromes (CAPS) stimulated further activation of caspase-1 extracellularly, as well as intracellularly after phagocytosis by surrounding macrophages. We found oligomeric ASC particles in the serum of patients with active CAPS but not in that of patients with other inherited autoinflammatory diseases. Our findings support a model whereby the NLRP3 inflammasome, acting as an extracellular oligomeric complex, amplifies the inflammatory response.