Induced Inflammasome Activation Research Articles

Protective effects of paeonol against cognitive impairment in lung diseases

Pulmonary inflammation may lead to neuroinflammation resulting in neurological dysfunction, and it is associated with a variety of acute and chronic lung diseases. Paeonol is a herbal phenolic compound with anti-inflammatory and anti-oxidative properties. The aim of this study is to understand the beneficial effects of paeonol on cognitive impairment, pulmonary inflammation and its underlying mechanisms. Pulmonary inflammation-associated cognitive deficit was observed in TNFα-stimulated mice, and paeonol mitigated the cognitive impairment by reducing the expressions of interleukin (IL)-1β, IL-6, and NOD-like receptor family pyrin domain-containing 3 (NLRP3) in hippocampus. Moreover, elevated plasma miR-34c-5p in lung-inflamed mice was also reduced by paeonol. Pulmonary inflammation induced by intratracheal instillation of TNFα in mice resulted in immune cells infiltration in bronchoalveolar lavage fluid, pulmonary edema, and acute fibrosis, and these inflammatory responses were alleviated by paeonol orally. In MH-S alveolar macrophages, tumor necrosis factor (TNF) α- and phorbol myristate acetate (PMA)-induced inflammasome activation was ameliorated by paeonol. In addition, the expressions of antioxidants were elevated by paeonol, and reactive oxygen species production was reduced. In this study, paeonol demonstrates protective effects against cognitive deficits and pulmonary inflammation by exerting anti-inflammatory and anti-oxidative properties, suggesting a powerful benefit as a potential therapeutic agent.

NKCC1-dependent K+ influx inhibits NLRP3 inflammasome activation and prevents intestinal epithelial cell injury

Background: Loss of function mutations in SLC12A2, the gene encoding the Na+-K+-2Cl− cotransporter (NKCC1) are associated with autoinflammatory and severe chronic inflammation in multiple organs. NLRP(s) are a family of cytosolic sensors that initiate the innate immune response. Once activated, NLRP(s) form inflammasomes, which are multi-protein complex that sense danger signals, damaged and infected cells. Assembly of the inflammasome mediates caspases-1 activation and the production of the proinflammatory cytokines IL-1β and IL-18 and initiates an inflammatory cell death process called pyroptosis. Aberrant activation of NLRP3 and other inflammasomes leads to a variety of autoimmune and chronic inflammatory diseases. Here we ask whether loss of NKCC1 function affects inflammasome activation in intestinal epithelial cells. Methods: Wild-type intestinal epithelial cells (IEC) and CRISPR/Cas9 NKCC1-deleted clones were tested for inflammasome activation. WT and NKCC1-deficient clones were treated with nigericin, ATP or ouabain to activate the inflammasome. Conditioned media, whole cell lysate and fixed cells were collected at different time points and analyzed by immunoblotting, immunofluorescence, and ELISA (to measure cytokine release). Inflammasome-induced pyroptosis was assessed by measuring LDH release in the supernatant and gasdermine D cleavage by immunoblotting. Results: Loss of NKCC1 function induces inflammasome activation by increasing IL-1β secretion in intestinal epithelial cells. Prolong inflammatory cytokine secretion led to increased pyroptosis-mediated cell death. Loss of NKCC1 mediates NLRP3 inflammasome activation by compromising K+ influx. NLRP3 agonists increase the interaction and localization of WNK1 and SPAK kinases with the NLRP3 inflammasome complex. Conclusions: NKCC1 regulates inflammatory cytokines secretion. Loss of NKCC1 inhibits K+ and Cl− inward fluxes, which in turn activate NLRP3 inflammasome. Loss of NKCC1 function is a previously unknown mechanism that causes aberrant inflammasome activation. GM118944 DK093501. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Downregulation of otulin induces inflammasome activation in neutrophilic asthma

Neutrophilic asthma (NA) is a severe asthma phenotype associated with steroid resistance and IL-1β overproduction; however, the exact mechanism remains unclear. Moreover, the dysfunction of tumor necrosis factor-alpha signaling pathway, a regulator of IL-1β production, was associated with the deficiency of OTU deubiquitinase with linear linkage specificity (otulin) in autoimmune patients. We hypothesized that otulin downregulation in macrophages (Mφ) could trigger Mφ activation via the nucleotide-binding domain, leucine-rich repeat, and pyrin domain-containing protein 3 (NLRP3) inflammasome signaling pathway. We assessed the expressions of otulin in blood monocyte subsets from NA patients and in alveolar Mφ from NA mice. Additionally, we evaluated the functional consequences of otulin deficiency in bone marrow-derived Mφ (BMDMs). The effects of inhibiting receptor-interacting protein kinase (RIPK)-1 and RIPK-3 on neutrophils and group 3 innate lymphoid cells (ILC3s) were assessed in vitro and in vivo. When comparing non-classical monocytes, a significant downregulation of otulin in the intracellular components was observed in NA patients when compared to healthy controls (P = 0.005). Additionally, isolated alveolar Mφ from the NA mice exhibited lower otulin expression compared to those from control mice. Following otulin knockdown in BMDMs, we observed spontaneous IL-1β production depending on NLRP3 inflammasome. Moreover, the infiltrated neutrophils and ILC3s were significantly decreased by combined treatment of RIPK-1 and RIPK-3 inhibitors through blocking IL-1β release in NA. Our findings suggest that IL-1β overproduction caused by a deficiency of otulin, an upstream triggering factor, could be a promising diagnostic and therapeutic target for NA.

Alternative autophagy dampens UVB-induced NLRP3 inflammasome activation in human keratinocytes

Sunlight exposure results in an inflammatory reaction of the skin commonly known as sunburn, which increases skin cancer risk. In particular, the ultraviolet B (UVB) component of sunlight induces inflammasome activation in keratinocytes to instigate the cutaneous inflammatory responses. Here, we explore the intracellular machinery that maintains skin homeostasis by suppressing UVB-induced inflammasome activation in human keratinocytes. We found that pharmacological inhibition of autophagy promoted UVB-induced NLRP3 inflammasome activation. Unexpectedly, however, gene silencing of Atg5 or Atg7, which are critical for conventional autophagy, had no effect, whereas gene silencing of Beclin1, which is essential not only for conventional autophagy but also for Atg5/Atg7-independent alternative autophagy, promoted UVB-induced inflammasome activation, indicating an involvement of alternative autophagy. We found that damaged mitochondria were highly accumulated in UVB-irradiated keratinocytes when alternative autophagy was inhibited, and they appear to be recognized by NLRP3. Overall, our findings indicate that alternative autophagy, rather than conventional autophagy, suppresses UVB-induced NLRP3 inflammasome activation through the clearance of damaged mitochondria in human keratinocytes, and illustrate a previously unknown involvement of alternative autophagy in inflammation. Alternative autophagy may be a new therapeutic target for sunburn and associated cutaneous disorders.

Tau induces inflammasome activation and microgliosis through acetylating NLRP3.

Alzheimer's disease (AD) and related Tauopathies are characterised by the pathologically hyperphosphorylated and aggregated microtubule-associated protein Tau, which is accompanied by neuroinflammation mediated by activated microglia. However, the role of Tau pathology in microglia activation or their causal relationship remains largely elusive. The levels of nucleotide-binding oligomerisation domain(NOD)-like receptor pyrin domain containing 3(NLRP3) acetylation and inflammasome activation in multiple cell models with Tau proteins treatment, transgenic mice with Tauopathy, and AD patients were measured by Western blotting and enzyme-linked immunosorbent assay. In addition, the acetyltransferase activity of Tau and NLRP3 acetylation sites were confirmed using the test-tube acetylation assay, co-immunoprecipitation, immunofluorescence (IF) staining, mass spectrometry and molecular docking. The Tau-overexpressing mouse model was established by overexpression of human Tau proteins in mouse hippocampal CA1 neurons through the adeno-associated virus injection. The cognitive functions of Tau-overexpressing mice were assessed in various behavioural tests, and microglia activation was analysed by Iba-1 IF staining and [18F]-DPA-714 positron emission tomography/computed tomography imaging. A peptide that blocks the interaction between Tau and NLRP3 was synthesised to determine the in vitro and in vivo effects of Tau-NLRP3 interaction blockade on NLRP3 acetylation, inflammasome activation, microglia activation and cognitive function. Excessively elevated NLRP3 acetylation and inflammasome activation were observed in 3xTg-AD mice, microtubule-associated protein Tau P301S (PS19) mice and AD patients. It was further confirmed that mimics of 'early' phosphorylated-Tau proteins which increase at the initial stage of diseases with Tauopathy, including TauT181E, TauS199E, TauT217E and TauS262E, significantly promoted Tau-K18 domain acetyltransferase activity-dependent NLRP3 acetylation and inflammasome activation in HEK293T and BV-2 microglial cells. In addition, Tau protein could directly acetylate NLRP3 at the K21, K22 and K24 sites at its PYD domain and thereby induce inflammasome activation in vitro. Overexpression of human Tau proteins in mouse hippocampal CA1 neurons resulted in impaired cognitive function, Tau transmission to microglia and microgliosis with NLRP3 acetylation and inflammasome activation. As a targeted intervention, competitive binding of a designed Tau-NLRP3-binding blocking (TNB) peptide to block the interaction of Tau protein with NLRP3 inhibited the NLRP3 acetylation and downstream inflammasome activation in microglia, thereby alleviating microglia activation and cognitive impairment in mice. In conclusion, our findings provide evidence for a novel role of Tau in the regulation of microglia activation through acetylating NLRP3, which has potential implications for early intervention and personalised treatment of AD and related Tauopathies.

P159 Inhibition of Caspase-1 signalling subtly affect the gut microbiota and reduces Adherent-invasive Escherichia coli (AIEC)-induced inflammasome activation

Abstract Background Inflammatory bowel disease (IBD), encompassing Crohn’s Disease and ulcerative colitis, is a chronic gastrointestinal disorder with increasing incidence worldwide. Microbial factors such as Adherent-invasive Escherichia coli (AIEC) have been suggested to play a significant role in the pathogenesis of IBD. The inflammasome, a multi-protein complex, senses environmental signals, including bacteria, resulting in the activation of caspase-1 leading to the subsequent maturation and secretion of IL-1β. In this study we examined the impact of AIEC on inflammasome activation and on the inhibition of Caspase-1 signaling in AIEC-colonised mice and on intestinal epithelial cells (IECs). Methods Human IECs (CaCO2Bee1 cells) were cultured for 3hr with AIEC (HM605 and its invasiveness mutant), washed with antibiotics and further cultured in full cell media for 13hrs, followed by analysis on cytokine and inflammasome gene expression and activation by qRT-PCR, ELISA, imaging and flow cytometry. To inhibit caspase-1 activation, cells were pre-treated for 1 hr with the caspase-1 inhibitor zVAD-fmk. To establish AIEC colonisation, C57BL/6 and IL-10-/- mice were treated with streptomycin for 24hrs followed by oral gavage of AIEC-HM605 (10-8 cfu/mL) and faecal and tissue samples were collected at 6 days post AIEC-infection. Caspase-1 inhibitor was orally gavaged every in mice. Faecal samples were prepared for 16S rRNA gene sequencing and colon tissue was analysed for cytokines and inflammasome markers by qRT-PCR and ELISA. Results IECs cultured with AIEC and AIEC-colonised C57BL/6 and IL-10-/- mice resulted in an increased expression and activation of both canonical (Caspase-1) and non-canonical inflammasome (caspase 4). The AIEC-induced inflammasome response was dependent on the invasiveness of AIEC. Treatment with a caspase-1 inhibitor reduced the AIEC-induced inflammasome markers in IECs and in C57BL/6 mice. No major changes in alpha or beta-diversity was detected in AIEC-colonised mice or upon Caspase-1 inhibition. AIEC-colonisation resulted in an increased abundance of Bacteroides and Alistipides and reduced abundance of Lachnospiraceae, alterations reversed by Caspase-1 inhibition. Conclusion We show that the invasion of AIEC in IECs and colonisation of murine intestine results in activation of caspase-1 inflammasome. AIEC colonisation provoke subtle microbiota alterations which are reversed by caspase-1 inhibition. In summary, our data supports a role of AIEC in IBD pathogenesis through the activation of caspase-1 inflammasome.

Flow-Induced Shear Stress Primes NLRP3 Inflammasome Activation in Macrophages via Piezo1.

The NLRP3 inflammasome is a crucial component of the innate immune system, playing a pivotal role in initiating and regulating the body's inflammatory response to various pathogens and cellular damage. Environmental stimuli, such as temperature, pH level, and nutrient availability, can influence the behavior and functions of innate immune cells, including immune cell activity, proliferation, and cytokine production. However, there is limited understanding regarding how mechanical forces, like shear stress, govern the intrinsic inflammatory reaction, particularly the activation of the NLRP3 inflammasome, and how shear stress impacts NLRP3 inflammasome activation through its capacity to induce alterations in gene expression and cytokine secretion. Here, we investigated how shear stress can act as a priming signal in NLRP3 inflammasome activation by exposing immortalized bone marrow-derived macrophages (iBMDMs) to numerous physiologically relevant magnitudes of shear stress before chemically inducing inflammasome activation. We demonstrated that shear stress of large magnitudes was able to prime iBMDMs more effectively for inflammasome activation compared to lower shear stress magnitudes, as quantified by the percentage of cells where ASC-CFP specks formed and IL-1β secretion, the hallmarks of inflammasome activation. Testing this in NLRP3 and caspase-1 knockout iBMDMs showed that the NLRP3 inflammasome was primarily primed for activation due to shear stress exposure. Quantitative polymerase chain reaction (qPCR) and a small-molecule inhibitor study mechanistically determined that shear stress regulates the NLRP3 inflammasome by upregulating Piezo1, IKKβ, and NLRP3. These findings offer insights into the mechanistic relationship among physiological shear stresses, inflammasome activation, and their impact on the progression of inflammatory diseases and their interconnected pathogenesis.

A Phase 2 Study of Canakinumab in Patients with Lower-Risk Myelodysplastic Syndromes or Chronic Myelomonocytic Leukemia

Background Myelodysplastic syndromes (MDS) lie on a continuum of myeloid neoplastic progression between clonal hematopoiesis (CH) and acute myeloid leukemia (AML). Interleukin 1 beta (IL-1β)-induced inflammasome activation is a mechanism of cancer initiation and progression. Given that IL-1β upregulation induces aberrant myeloid differentiation of hematopoietic stem and progenitor cells (HSPCs), we hypothesize that IL-1β plays a critical role in MDS pathogenesis, and targeting IL-1β signaling will overcome aberrant MDS HSPCs' myeloid skewing. Here, we report final results of a phase 2 study evaluating the safety and efficacy of canakinumab, an IL-1β inhibitor, in patients with lower-risk MDS or chronic myelomonocytic leukemia (CMML). Methods We conducted a single center, open-label phase 2 trial at The University of Texas MD Anderson Cancer Center (Houston, TX, USA). Patients aged ≥ 18 with IPSS-R ≤ 3.5 MDS/CMML who have previously been treated were included in the study. Patients received 300 mg of canakinumab on day 1 of a 4-week cycle. The primary objective was to determine the clinical activity of canakinumab in MDS/CMML patients. Response was assessed using the modified International Working Group 2006 criteria for MDS (Cheson et al. 2006). Secondary objectives were to evaluate the safety profile including rate of transfusion independence (TI), duration of response (DoR), progression-free survival (PFS), and overall survival (OS). All patients who received 1 dose of study drug were included in the analyses. This study is registered with ClinicalTrials.gov (NCT04239157). Findings Between August 2020 and May 2023, 27 patients were enrolled in the phase 2 portion of this study, and 2 patients failed screening. The median age was 74 years with 15 (60%) male patients; 20 patients (80%) were post-hypomethylating agent failure with a median number of prior lines of therapy of 2 (1-5). Transfusion dependency was observed in 24 patients (96%) prior to canakinumab initiation. Fourteen (56%) patients showed normal karyotype. IPSS-R stratification revealed an intermediate-2 risk in 12 (48%) patients and a high risk in one (4%). The most common mutations were SF3B1 (40%), TET2 (32%), DNMT3A (28%), and RUNX1 (24%). IPSS -M risk score was calculated for 24 cases showing one (4%) very low (VL), four (16%) low (L), 14 (16%) moderate low (ML), eight (32%) moderate high (MH), five (20%) high (H), and two (8%) very high (VH) risk categories. Canakinumab was well tolerated and no drug-related toxicities were observed. One death due to sepsis, which was deemed not treatment-related, occurred on the study drug. Out of 23 evaluable patients, the overall response rate was 17·4%, with erythroid and platelet hematological improvement (HI-E and HI-P, respectively) confirmed in 3 (13.0%) patients and 1 (4.3%) patient, respectively. Thirteen patients had stable disease (56·5%) and 6 (26·1%) progressed during therapy, 1 of which transformed to AML (Fig. 1). TI was achieved in 3 patients (median DoR 8·53 months (95% CI 0·41-16·1) and 2 of them maintained TI for over 12 months. With a median follow-up time of 22·6 months (95% CI 15·0-29·4), median OS was 17·3 months (95% CI 14·3-not estimable). We performed separate univariate analyses to evaluate any associations between the IPSS-M and OS/PFS. The median OS in patients with higher-risk MDS by IPSS-M (MH, H, VH) was 15·0 months vs 29·4 months in the lower-risk disease by IPSS-M (VL, L, ML) group (p=0·12). Interestingly, statistically significant findings were observed with 1-year PFS when stratifying patients into higher vs lower risk MDS by IPSS-M (64·3% vs 100·0%, respectively; p=0·022, Fig. 2). Conclusion In this cohort of MDS patients who had experienced multiple lines of prior therapy and exhibited high-molecular-complexity, canakinumab showed limited efficacy (HI 17.3%). Nevertheless, canakinumab showed a good safety profile and yielded sustained long-term responses in patients with single somatic driver mutation in TET2 or DNMT3A. This suggests that clonal complexity, and therefore disease burden, may be a determining factor in response to canakinumab. Therefore, we have amended the protocol of a phase 1/2 clinical trial evaluating the safety and activity of canakinumab in clonal cytopenia of unknown significance (CCUS) and low-molecular complexity MDS patients. Our results will clarify the role of IL-1β signaling in MDS initiation and progression.

Macrophage IL-1β contributes to tumorigenesis through paracrine AIM2 inflammasome activation in the tumor microenvironment.

Intracellular recognition of self and non-self -nucleic acids can result in the initiation of effective pro-inflammatory and anti-tumorigenic responses. We hypothesized that macrophages can be activated by tumor-derived nucleic acids to induce inflammasome activation in the tumor microenvironment. We show that tumor conditioned media (CM) can induce IL-1β production, indicative of inflammasome activation in primed macrophages. This could be partially dependent on caspase 1/11, AIM2 and NLRP3. IL-1β enhances tumor cell proliferation, migration and invasion while coculture of tumor cells with macrophages enhances the proliferation of tumor cells, which is AIM2 and caspase 1/11 dependent. Furthermore, we have identified that DNA-RNA hybrids could be the nucleic acid form which activates AIM2 inflammasome at a higher sensitivity as compared to dsDNA. Taken together, the tumor-secretome stimulates an innate immune pathway in macrophages which promotes paracrine cancer growth and may be a key tumorigenic pathway in cancer. Broader understanding on the mechanisms of nucleic acid recognition and interaction with innate immune signaling pathway will help us to better appreciate its potential application in diagnostic and therapeutic benefit in cancer.

Endothelial P2X7 promotes venous thromboembolism

Venous thromboembolism (VTE) affects 117 people per 100,000 each year and is an important cause of morbidity and mortality. VTE can lead to 1) death through pulmonary embolism, 2) the post-thrombotic syndrome, or 3) pulmonary sequelae that dramatically impair quality of life. Thrombosis has long been characterized by the Virchow's triad encompassing hypercoagulability, venous stasis and vascular wall damage. However, inflammatory pathways are now well recognized mechanisms involved in the physiopathology of venous thromboembolism. Endothelial cells play critical roles in regulating immune functions in response to PAMPs and DAMPs. During inflammation, adenosine triphosphate (ATP) is released in the extracellular compartment and is recognized as a danger signal by endothelial cells. ATP can interact with the CD39/CD73 system involved in the metabolism of ATP into AMP. Recent data indicated that the CD39/CD73 system might protect against venous thrombosis by downregulating the NLRP3 inflammasome. ATP can also interact with the P2X7 receptor involved in a wide range of responses including NLRP3 inflammasome activation leading to IL1β production. However, the role of P2X7 receptor in VTE is unknown. To determine how the endothelial P2X7 receptor contributes to venous thromboembolism. After pretreatment with TNFα, HUVECs were incubated with BzATP alone or with thrombin. Immunofluorescence, western blot and real-time quantitative PCR analyses were used to study P2X7 expression in endothelial cells, activation of p38 and NFκB signaling pathways and gene expression, respectively. We confirmed that HUVECs expressed P2X7 receptor in vitro and in vivo after induction of venous thrombosis in an experimental model. BzATP and thrombin induced the activation of p38 and NFκB signaling pathways. Interestingly, this was associated with inflammasome assembly and increased IL1β expression. In addition, ICAM-1 and VCAM-1 expression was increased, while thrombomodulin expression was decreased. Our data suggest that ATP released in the extracellular space during venous thrombosis induced inflammasome activation in endothelial cell through P2X7 activation. P2X7 might have a pro-thrombotic role exacerbating venous thromboembolism.

A22 USING INTESTINAL ORGANOIDS TO CHARACTERIZE THE NAIP-NLRC4 INFLAMMASOME RESPONSE IN THE INTESTINAL EPITHELIUM

Abstract Background The NAIP-NLRC4 inflammasome is an important innate defence mechanism in intestinal epithelial cells (IECs) that protects the gut from invasive pathogens. NAIP-NLRC4 activation triggers pyroptotic cell death, releases active interleukin (IL)-18, and promotes the expulsion of infected enterocytes. Dysregulated inflammasomes result in exaggerated inflammation of the intestinal mucosa which is characterisitic of inflammatory bowel disease (IBD). There is increasing evidence that inflammasomes in IEC behave differently than in immune cells. However, a majority of inflammasome pathway characterization is studied immune cells. In addition, NAIP-NLRC4 activity in macrophages has been shown to be important for regulating the expression of nitric oxide (Nos2). The potential transcriptomic impact of NAIP-NLRC4 activity in IECs has yet to be explored. Purpose We will use intestinal orgnaoids to systematically characterize the NAIP-NLRC4 inflammasome pathway and identify the transcriptomic impact of NAIP-NLRC4 activity in IECs. To better define the role of NAIP-NLRC4 activity during a physiological infection, we have developed a novel ex vivo model of Shigella infection in 3D organoids. Method Organoids were derived from the ileal crypts of wild type (WT) and Nlrc4-/-, Casp1-/-, Pycard-/-, or Tlr5-/- mice and stimulated with Pam3CSk4, LPS, or flagellin. Inflammasome activation was assessed by Western blot (WB) and propidium iodide uptake. WT and Nlrc4-/- organoids were infected with WT or a non-invasive Shigella mutant and the inflammasome response was evaluated by WB and a colony forming unit assay. WT and Nlrc4-/- organoids were stimulated with flagellin and gene expression was assessed by RT-qPCR. Result(s) Basolateral organoid stimulation with bacterial ligands revealed a novel response of IECs to bacterial flagellin that results in pyroptosis and IL-18 processing. Basolateral internalization of flagellin occurred in a TLR5-independent manner. Inflammasome activation by flagellin was fully abrogated in Nlrc4-/- and Casp1-/- organoids while only IL-18 processing was affected in Pycard-/- organoids. Infection with only WT Shigella induced inflammasome activation in an NLRC4-dependent manner. Interestingly, flagellin stimulation of WT but not Nlrc4-/- organoids led to increased expression of Nos2. Furthermore, Nlrc4-/- organoids had significantly lower expression levels of cytokine genes Ccl20, Cxcl1, and Tnf following inflammasome activation. Conclusion(s) Our study demonstrates an integral role for epithelial NAIP-NLRC4 inflammasomes in the response to bacterial flagellin and Shigella infection. We have uncovered a novel response of IECs to basolateral flagellin stimulation and revealed that NAIP-NLRC4 is important for the transcriptional regulation of inflammatory genes. Further work will examine how NAIP-NLRC4 activation controls inflammation and epithelial integrity by analyzing the NLRC4-dependent transcriptome and the effects on cell proliferation, differentiation, and barrier integrity. Please acknowledge all funding agencies by checking the applicable boxes below CCC, CIHR Disclosure of Interest None Declared

Dual Role of Mitogen-Activated Protein Kinase 8 Interacting Protein-1 in Inflammasome and Pancreatic β-Cell Function

Inflammasomes have been implicated in the pathogenesis of type 2 diabetes (T2D). However, their expression and functional importance in pancreatic β-cells remain largely unknown. Mitogen-activated protein kinase 8 interacting protein-1 (MAPK8IP1) is a scaffold protein that regulates JNK signaling and is involved in various cellular processes. The precise role of MAPK8IP1 in inflammasome activation in β-cells has not been defined. To address this gap in knowledge, we performed a set of bioinformatics, molecular, and functional experiments in human islets and INS-1 (832/13) cells. Using RNA-seq expression data, we mapped the expression pattern of proinflammatory and inflammasome-related genes (IRGs) in human pancreatic islets. Expression of MAPK8IP1 in human islets was found to correlate positively with key IRGs, including the NOD-like receptor (NLR) family pyrin domain containing 3 (NLRP3), Gasdermin D (GSDMD) and Apoptosis-associated speck-like protein containing a CARD (ASC), but correlate inversely with Nuclear factor kappa β1 (NF-κβ1), Caspase-1 (CASP-1), Interleukin-18 (IL-18), Interleukin-1β (IL-1β) and Interleukin 6 (IL-6). Ablation of Mapk8ip1 by siRNA in INS-1 cells down-regulated the basal expression levels of Nlrp3, NLR family CARD domain containing 4 (Nlrc4), NLR family CARD domain containing 1 (Nlrp1), Casp1, Gsdmd, Il-1β, Il-18, Il-6, Asc, and Nf-κβ1 at the mRNA and/or protein level and decreased palmitic acid (PA)-induced inflammasome activation. Furthermore, Mapk8ip1-silened cells substantially reduced reactive oxygen species (ROS) generation and apoptosis in palmitic acid-stressed INS-1 cells. Nonetheless, silencing of Mapk8ip1 failed to preserve β-cell function against inflammasome response. Taken together, these findings suggest that MAPK8IP1 is involved in regulating β-cells by multiple pathways.

Clinical and Biological Effects of Canakinumab in Lower-Risk Myelodysplastic Syndromes (MDS): Results from a Phase 2 Clinical Trial

Interleukin 1 beta (IL-1B) induced inflammasome activation is a mechanism of cancer initiation and progression (Schroder K, Cell, 2010). Our previous studies (Ganan-Gomez I, Blood 2021) showed that IL-1B was one of the most overexpressed pro-inflammatory cytokines in the hematopoietic stem and progenitor cells (HSPCs) of patients with clonal cytopenia of undetermined significance (CCUS) carrying DNMT3a and TET2 mutations. Given that IL-1B upregulation induces aberrant myeloid differentiation of HSPCs (Pietras EM, Nat Cell Biol., 2016), we hypothesize that IL-1B plays a critical role in MDS pathogenesis, and that targeting IL-1B signaling overcomes aberrant MDS HSPCs' myeloid skewing. To test this hypothesis, we designed a clinical trial to evaluate the IL-1B inhibitor canakinumab's (NCT04239157) safety profile and its clinical and biological effects in patients with lower-risk MDS. Patients and Methods: Patients aged ≥ 18 with IPSS-R ≤ 3.5 MDS, normal renal and hepatic functions, and performance status, who had previously received at least 1 line of other therapy were included in the study (Table1). Patients received 300 mg of canakinumab on day 1 of a 4-week cycle. Bone marrow (BM) samples were obtained before therapy and after 1, 2, and 4 cycles of therapy during the trial and after every 3 or 4 cycles of therapy thereafter. Single-cell RNA sequencing (scRNA-seq; 10X Genomics) analyses were performed in lineage negative (Lin-)CD34+ HSPCs and mononuclear cells (MNCs) isolated at different therapy timepoints from the BM of 1 patient who achieved hematological improvement after canakinumab treatment, 2 patients who achieved stable disease, and 2 patients who did not respond to this therapy. The clinical characteristics of the 5 patients whose samples were analyzed by scRNA-seq analyses are included in Table 1. Results: Twenty-six patients have been enrolled in the clinical trial. Patients' clinical characteristics are included in Table 1. Twenty-three patients were evaluable for response. Among the 22 out of the 23 patients who were transfusion dependent at baseline, one achieved transfusion independence, and another had platelet count improvement. Three out of the 23 had progressive disease, and 18 had stable disease. Treatment was well tolerated with no severe adverse events. One patient died of sepsis unrelated to therapy. Overall, we did not observe any improvement in the blast counts and cytogenetic and/or mutational burden. To evaluate the biological effects of canakinumab, we performed scRNA-seq of Lin-CD34+ HSPCs (Figure 1) and MNCs at therapy baseline and after 2, 4, 5, 8, and 11 cycles of treatment. Consistent with our initial hypotheses, these analyses revealed that canakinumab decreased the frequencies of hematopoietic stem cells (HSCs) and multipotent myeloid progenitors (MPPs). Differential analysis between the samples showed that HSCs and MPPs significantly downregulated the expression of genes involved in cytokine signaling (e.g., IL-1B, CXCL2, and CXCL8), TNFα-induced activation of NF-κB, and hypoxia pathways, which demonstrated efficient target engagement of canakinumab on MDS cells. Similar results were also observed in downstream monocytes and B cells. In contrast, we did not observe any significant change in the HSPC architecture in the BM of patients with stable disease or whose disease did not respond to canakinumab. Consistent with our previous results in early stage MDS, the patient who achieved hematological improvement after canakinumab therapy was the only one among the cohort who carried a single mutation affecting DNMT3A (Table1). Conclusion. This study demonstrates that canakinumab is safe in MDS and efficiently targets IL-1B signaling. However, the biological effect of canakinumab has not yet translated in clinical responses in the subset of patients enrolled in this clinical trial. The study will accrue transfusion independent patients and patients with early stage MDS as CCUS. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A novel cysteine protease inhibitor in Baylisascaris schroederi migratory larvae regulates inflammasome activation through the TLR4–ROS–NLRP3 pathway

BackgroundGiant pandas (Ailuropoda melanoleuca) are the obligate host of the parasitic roundworm Baylisascaris schroederi. The infection of giant pandas with B. schroederi is very common. At present, little is known about the mechanism of immune interaction between B. schroederi and the host. As an important component of innate immunity, the NOD-like receptor 3 (NLRP3) inflammasome plays an important role in host immune response and the occurrence and development of infectious diseases.MethodsWe analyzed the regulation of NLRP3 inflammasome activation in monocyte-derived macrophages (MDMs) by the recombinant B. schroederi migratory larvae cysteine protease inhibitor rBsCPI-1, knowing from a previous study that the CPI-1 is highly expressed in B. schroederi migratory larvae. We first determined the effects of rBsCPI-1 and excretory–secretory products of B. schroederi migratory larvae on cell proliferation using the CCK-8 and LDH release assays. We then analyzed NLRP3 inflammasome activation, pyroptosis and pro-inflammatory cytokine release by quantitative-PCR, western blotting and enzyme-linked immunosorbent assay. The signaling pathway of rBsCPI-1 to activate NLRP3 inflammasomes was analyzed in activation and inhibition experiments. Finally, the effects of rBsCPI-1 on inflammasome activation in mice immunized with rBsCPI-1 were analyzed.ResultsThe activation and inhibition experiments revealed that rBsCPI-1 induced inflammasome activation through the TLR4–ROS–NLRP3 signaling pathway, with reactive oxygen species (ROS) not only functioning as an activator of the NLRP3 inflammasome, but also an activation product of the NLRP3 inflammasome. rBsCPI-1 promoted the activation and assembly of the NLRP3 inflammasome, which further converted the pro-inflammatory cytokines interleukin (IL)-1β and IL-18 into mature active forms. At the same time, caspase-1 cleaved gasdermin D to trigger cell pyroptosis. The results of animal immunization experiments further confirmed that rBsCPI-1 could induce the activation of the NLRP3 inflammasome.ConclusionsrBsCPI-1 activates the inflammasome through the TLR4–ROS–NLRP3 signaling pathway and further induces the pyroptosis of MDMs and release of pro-inflammatory factors IL-1β and IL-18, thus promoting the occurrence and development of the inflammatory response in the host.Graphical abstract

Development of a cell line-based in vitro assay for assessment of Diphtheria, Tetanus and acellular Pertussis (DTaP)-induced inflammasome activation

Safety and potency assessment for batch release testing of established vaccines still relies partly on animal tests. An important avenue to move to batch release without animal testing is the consistency approach. This approach is based on thorough characterization of the vaccine to identify critical quality attributes that inform the use of a comprehensive set of non-animal tests to release the vaccine, together with the principle that the quality of subsequent batches follows from their consistent production. Many vaccine antigens are by themselves not able to induce a protective immune response. The antigens are therefore administered together with adjuvant, most often by adsorption to aluminium salts. Adjuvant function is an important component of vaccine potency, and an important quality attribute of the final product. Aluminium adjuvants are capable of inducing NLRP3 inflammasome activation. The aim of this study was to develop and evaluate an in vitro assay for NLRP3 inflammasome activation by aluminium-adjuvanted vaccines. We evaluated the effects of Diphtheria-Tetanus-acellular Pertussis combination vaccines from two manufacturers and their respective adjuvants, aluminium phosphate (AP) and aluminium hydroxide (AH), in an in vitro assay for NLRP3 inflammasome activation. All vaccines and adjuvants tested showed a dose-dependent increase in IL-1β production and a concomitant decrease in cell viability, suggesting NLRP3 inflammasome activation. The results were analysed by benchmark dose modelling, showing a similar 50% effective dose (ED50) for the two vaccine batches and corresponding adjuvant of manufacturer A (AP), and a similar ED50 for the two vaccine batches and corresponding adjuvant of manufacturer B (AH). This suggests that NLRP3 inflammasome activation is determined by the adjuvant only. Repeated freeze-thaw cycles reduced the adjuvant biological activity of AH, but not AP. Inflammasome activation may be used to measure adjuvant biological activity as an important quality attribute for control or characterization of the adjuvant.

Clostridium difficile Induced Inflammasome Activation and Coagulation Derangements.

C. difficile enterocolitis (CDAC) is the most common hospital infection, burdened by an increased incidence of coagulation-related complications such as deep vein thrombosis (DVT) and disseminated intravascular coagulation (DIC) as well as a significant sepsis-related mortality. In this review, we analyzed the available data concerning the correlation between coagulation complications related to C. difficile infection (CDI) and inflammasome activation, in particular the pyrin-dependent one. The little but solid available preclinical and clinical evidence shows that inflammasome activation increases the risk of venous thromboembolism (VTE). As proof of this, it has been observed that in vitro inhibition of the molecules (e.g., tissue factor) mainly involved in coagulation activation could block the process. In vivo studies show that it could be possible to reduce the incidence of complications associated with C. difficile infection (CDI) and mortality due to a state of hypercoagulability. A personalized therapeutic approach to reduce the inflammatory activity and prevent thromboembolic complications could be preliminarily defined to reduce mortality.

Somatic gene mutations expose cytoplasmic DNA to co-opt the cGAS/STING/NLRP3 axis in myelodysplastic syndromes.

NLRP3 inflammasome and IFN-stimulated gene (ISG) induction are key biological drivers of ineffective hematopoiesis and inflammation in myelodysplastic syndromes (MDSs). Gene mutations involving mRNA splicing and epigenetic regulatory pathways induce inflammasome activation and myeloid lineage skewing in MDSs through undefined mechanisms. Using immortalized murine hematopoietic stem and progenitor cells harboring these somatic gene mutations and primary MDS BM specimens, we showed accumulation of unresolved R-loops and micronuclei with concurrent activation of the cytosolic sensor cyclic GMP-AMP synthase. Cyclic GMP-AMP synthase/stimulator of IFN genes (cGAS/STING) signaling caused ISG induction, NLRP3 inflammasome activation, and maturation of the effector protease caspase-1. Deregulation of RNA polymerase III drove cytosolic R-loop generation, which upon inhibition, extinguished ISG and inflammasome response. Mechanistically, caspase-1 degraded the master erythroid transcription factor, GATA binding protein 1, provoking anemia and myeloid lineage bias that was reversed by cGAS inhibition in vitro and in Tet2–/– hematopoietic stem and progenitor cell–transplanted mice. Together, these data identified a mechanism by which functionally distinct mutations converged upon the cGAS/STING/NLRP3 axis in MDS, directing ISG induction, pyroptosis, and myeloid lineage skewing.

620 Inflammasome activation in human keratinocytes and mouse epidermis by ultraviolet radiation

Ultraviolet B radiation (UVB) is a ubiquitous environmental toxin that causes extensive skin damage resulting in inflammation, photoaging and cancer. UVB is known to induce inflammasome activation and a sterile inflammation associated with cytokine release (e.g. IL-1β, IL-18) and immune cell infiltration. We developed circularly permutated luciferase reporters for caspase-1 activation to assess inflammasome activation by UVB in vitro and in vivo. HaCaT keratinocytes transduced with caspase-1 reporters encoding either the caspase-7 or Gasdermin D cleavage sequence became active after exposure to UVB, with maximum caspase-1 activation 9 hours post-UVB with 15 mJ/cm2.

Activating cGAS–STING axis contributes to neuroinflammation in CVST mouse model and induces inflammasome activation and microglia pyroptosis

BackgroundNeuroinflammation-induced injury is intimately associated with poor prognosis in patients with cerebral venous sinus thrombosis (CVST). The cyclic GMP-AMP synthase–stimulator of interferon gene (cGAS–STING) axis is a cytoplasmic double-stranded DNA (dsDNA) sensing pathway has recently emerged as a crucial mediator of neuroinflammation in ischemic stroke. However, the role of the cGAS–STING pathway in modulating post-CVST inflammation and the underlying mechanisms involved remain unclear.MethodsA CVST model was induced by ferric chloride in male C57BL/6J mice. The selective cGAS inhibitor RU.521, STING agonist 2′3′-cGAMP, and STING siRNA were delivered by intranasal administration or intraventricular injection. Post-CVST assessments included rotarod test, TUNEL staining, Fluoro-Jade C staining, dihydroethidium staining, western blotting, qPCR, immunofluorescence, immunohistochemistry, ELISA and flow cytometry.ResultscGAS, STING, NLRP3 and GSDMD were significantly upregulated after CVST and mostly in the microglia of the mouse brain. CVST triggered the release of dsDNA into the cytoplasm and elicited an inflammatory response via activating the cGAS–STING axis. RU.521 decreased the levels of 2′3′-cGAMP, STING and downstream inflammatory cytokines, and suppressed the expressions of NLRP3 inflammasome and pyroptosis-pertinent components containing cleaved caspase-1, GSDMD, GSDMD-C, pro- and cleaved IL-1β, and cleaved IL-1β/pro-IL-1β. Besides, RU.521 treatment also reduced oxidative stress, lessened the numbers of microglia and neutrophils, and ameliorated neuronal apoptosis, degeneration along with neurological deficits post-CVST. 2′3'-cGAMP delivery enhanced the expressions of STING and related inflammatory mediators, NLRP3 inflammasome and pyroptosis-relevant proteins, whereas these alterations were significantly abrogated by the silencing of STING by siRNA.ConclusionsOur data demonstrate that repression of the cGAS–STING pathway diminishes the neuroinflammatory burden of CVST and highlight this approach as a potential therapeutic tactic in CVST-mediated pathologies.

Clostridioides difficile surface layer protein triggered inflammasome activation is mediated by membrane lipid raft

Clostridioides difficile is the leading cause of nosocomial infectious diarrhea, with clinical symptoms ranging from mild diarrhea to pseudomembranous colitis and toxic megacolon. Despite the use of vancomycin and fidaxomicin as standard drugs for the treatment of C. difficileinfection (CDI), clinical relapse rates remain high. Therefore, new alternative therapeutics to treat CDI are urgently required. Surface layer proteins (SLPs) are the most abundant proteins in the C. difficile cell wall, suggesting that they might involve in immune recognition. Here, we found SLPs as well as C. difficile induced inflammasome activation. In addition, the cholesterol‐rich microdomains, lipid rafts, on the cell membrane are thought to be crucial for bacterial adhesion and signal transduction. We demonstrated that lipid rafts participated in C. difficile SLPs binding to the cell membrane. Furthermore, both binding ability and inflammasome activation induced by SLP were abrogated with membrane cholesterol depletion by methyl‐β‐cyclodextrin (MβCD). The coalescence of SLPs in the cholesterol‐rich microdomains was confirmed in C. difficile‐infected cells. Our results demonstrate that SLPs recruit the lipid rafts, which may be a key step for C. difficile colonization and inducing inflammasome activation.