Inhibition Of IL-1β Production Research Articles

The BK Channel Limits the Pro-Inflammatory Activity of Macrophages.

Macrophages play a crucial role in the innate immune response, serving as key effector cells in the defense against pathogens. Although the role of the large-conductance voltage and calcium-activated potassium channel, also known as the KCa1.1 or BK channel, in regulating neurotransmitter release and smooth muscle contraction is well known, its potential involvement in immune regulation remains unclear. We employed BK-knockout macrophages and noted that the absence of a BK channel promotes the polarization of macrophages towards a pro-inflammatory phenotype known as M1 macrophages. Specifically, the absence of the BK channel resulted in a significant increase in the secretion of the pro-inflammatory cytokine IL-6 and enhanced the activity of extracellular signal-regulated kinases 1 and 2 (Erk1/2 kinases), Ca2+/calmodulin-dependent protein kinase II (CaMKII), and the transcription factor ATF-1 within M1 macrophages. Additionally, the lack of the BK channel promoted the activation of the AIM2 inflammasome without affecting the activation of the NLRC4 and NLRP3 inflammasomes. To further investigate the role of the BK channel in regulating AIM2 inflammasome activation, we utilized BK channel inhibitors, such as paxilline and iberiotoxin, along with the BK channel activator NS-11021. Pharmacological inactivation of the BK channel increased, and its stimulation inhibited IL-1β production following AIM2 inflammasome activation in wild-type macrophages. Moreover, wild-type macrophages displayed increased calcium influx when activated with the AIM2 inflammasome, whereas BK-knockout macrophages did not due to the impaired extracellular calcium influx upon activation. Furthermore, under conditions of a calcium-free medium, IL-1β production following AIM2 inflammasome activation was increased in both wild-type and BK-knockout macrophages. This suggests that the BK channel is required for the influx of extracellular calcium in macrophages, thus limiting AIM2 inflammasome activation. In summary, our study reveals a regulatory role of the BK channel in macrophages under inflammatory conditions.

Pharmacology of a Potent and Novel Inhibitor of the NOD-Like Receptor Pyrin Domain-Containing Protein 3 (NLRP3) Inflammasome that Attenuates Development of Nonalcoholic Steatohepatitis and Liver Fibrosis.

The NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome is a multiprotein complex and component of the innate immune system that is activated by exogenous and endogenous danger signals to promote activation of caspase-1 and the maturation and release of the proinflammatory cytokines interleukin (IL)-1β and IL-18. Inappropriate activation of NLRP3 has been implicated in the pathophysiology of multiple inflammatory and autoimmune diseases, including cardiovascular disease, neurodegenerative diseases, and nonalcoholic steatohepatitis (NASH), thus increasing the clinical interest of this target. We describe in this study the preclinical pharmacologic, pharmacokinetic, and pharmacodynamic properties of a novel and highly specific NLRP3 inhibitor, JT001 (6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazine-3-sulfonylurea). In cell-based assays, JT001 potently and selectively inhibited NLRP3 inflammasome assembly, resulting in the inhibition of cytokine release and the prevention of pyroptosis, a form of inflammatory cell death triggered by active caspase-1. Oral administration of JT001 to mice inhibited IL-1β production in peritoneal lavage fluid at plasma concentrations that correlated with mouse in vitro whole blood potency. Orally administered JT001 was effective in reducing hepatic inflammation in three different murine models, including the Nlrp3A350V /+CreT model of Muckle-Wells syndrome (MWS), a diet-induced obesity NASH model, and a choline-deficient diet-induced NASH model. Significant reductions in hepatic fibrosis and cell damage were also observed in the MWS and choline-deficient models. Our findings demonstrate that blockade of NLRP3 attenuates hepatic inflammation and fibrosis and support the use of JT001 to investigate the role of NLRP3 in other inflammatory disease models. SIGNIFICANCE STATEMENT: Persistent inflammasome activation is the consequence of inherited mutations of NLRP3 and results in the development of cryopyrin-associated periodic syndromes associated with severe systemic inflammation. NLRP3 is also upregulated in nonalcoholic steatohepatitis, a metabolic chronic liver disease currently missing a cure. Selective and potent inhibitors of NLRP3 hold great promise and have the potential to overcome an urgent unmet need.

Clostridioides difficile aggravates dextran sulfate solution (DSS)-induced colitis by shaping the gut microbiota and promoting neutrophil recruitment

ABSTRACT Clostridioides difficile is a pathogen contributing to increased morbidity and mortality of patients with inflammatory bowel disease (IBD). To determine how C. difficile affects the severity of colitis, we constructed a dextran sulfate solution-induced colitis model challenged with C. difficile. Without antibiotic administration, C. difficile led to transient colonization in mice with colitis, but still significantly enhanced disease severity as assessed by weight loss, histopathological damages, and inflammatory cytokine concentrations. Because this effect is independent of toxin production as shown by infection with a non-toxigenic strain, we focused on changes in the gut microbiota. The microbiota altered by C.difficile, featured with reduced proportions of g_Prevotellaceae_UCG-001 and g_Muribaculaceae, were confirmed to contribute to disease severity in colitis mice via fecal microbiota transplantations. The inflamed colon showed neutrophil accumulation by flow cytometric analysis and myeloperoxidase immunochemical staining. There was enrichment of upregulated genes in leukocyte chemotaxis or migration as shown by RNA sequencing analysis. The isolated neutrophils from C. difficile-infected mice with colitis showed a robust migratory ability and had enhanced expression of cytokines and chemokines. We observed a detrimental role of neutrophils in the progress of disease by hindering neutrophil recruitment with the CXCR2 inhibitor SB225002. Furthermore, neutrophil recruitment appeared to be regulated by interleukin (IL)-1β, as inhibition of IL-1β production by MCC950 markedly ameliorated inflammation with decreased neutrophil accumulation and neutrophil-derived chemokine expression. In conclusion, our study provides information on the complicated interaction between microbiota and immune responses in C. difficile-induced inflammation in mice with colitis. Our findings could help determine potential therapeutic targets for patients with IBD concurrent with C. difficile infection.

Leishmanicidal and immunomodulatory properties of Brazilian green propolis extract (EPP-AF®) and a gel formulation in a pre-clinical model.

Leishmaniasis is a widespread group of neglected vector-borne tropical diseases that possess serious therapeutic limitations. Propolis has been extensively used in traditional medical applications due to its range of biological effects, including activity against infectious agents. Here we evaluated the leishmanicidal and immunomodulatory properties of Brazilian green propolis extract (EPP-AF®) and a gel formulation incorporating EPP-AF®, in both in vitro and in vivo models of Leishmania amazonensis infection. Propolis extract, obtained from a standardized blend following hydroalcoholic extraction, showed the characteristic fingerprint of Brazilian green propolis as confirmed by HPLC/DAD. A carbopol 940 gel formulation was obtained containing propolis glycolic extract at 3.6% w/w. The release profile, assessed using the Franz diffusion cell protocol, demonstrated a gradual and prolonged release of p-coumaric acid and artepillin C from the carbomer gel matrix. Quantification of p-coumaric acid and artepillin C in the gel formulation over time revealed that p-coumaric acid followed the Higuchi model, dependent on the disintegration of the pharmaceutical preparation, while artepillin C followed a zero-order profile with sustained release. In vitro analysis revealed the ability of EPP-AF® to reduce the infection index of infected macrophages (p < 0.05), while also modulating the production of inflammatory biomarkers. Decreases in nitric oxide and prostaglandin E2 levels were observed (p < 0.01), suggesting low iNOS and COX-2 activity. Furthermore, EPP-AF® treatment was found to induce heme oxygenase-1 antioxidant enzyme expression in both uninfected and L. amazonensis-infected cells, as well as inhibit IL-1β production in infected cells (p < 0.01). ERK-1/2 phosphorylation was positively correlated with TNF-α production (p < 0.05), yet no impact on parasite load was detected. In vivo analysis indicated the effectiveness of topical treatment with EPP-AF® gel alone (p < 0.05 and p < 0.01), or in combination with pentavalent antimony (p < 0.05 and p < 0.001), in the reduction of lesion size in the ears of L. amazonensis-infected BALB/c mice after seven or 3weeks of treatment, respectively. Taken together, the present results reinforce the leishmanicidal and immunomodulatory effects of Brazilian green propolis, and demonstrate promising potential for the EPP-AF® propolis gel formulation as a candidate for adjuvant therapy in the treatment of Cutaneous Leishmaniasis.

Modulation of Neutrophil Function by Recombinant Human IgG1 Fc Hexamer in the Endogenous K/BxN Mouse Model of Rheumatoid Arthritis

Introduction: Neutrophils are a pivotal cell type in the K/BxN mouse model of rheumatoid arthritis and play an essential role in the progression of the arthritis. They are readily activated by immune complexes (ICs) via their FcγRs to release IL-1β in addition to other cytokines, which are inducing cartilage destruction. Neutrophils also release neutrophil-active chemokines to recruit themselves in an autocrine manner to perpetuate tissue destruction. FcγR-expression on neutrophils is of crucial importance for the recognition of ICs. Methods: In this study, due to its high avidity for binding to FcγRs, we investigated the potential anti-inflammatory effect of a recombinant IgG1 Fc hexamer (rFc-µTP-L309C) on neutrophils in the K/BxN mouse model of endogenously generated chronic arthritis. 200 mg/kg rFc-µTP-L309C and human serum albumin (HSA), used as controls, were administered subcutaneously every other day. Mouse ankle joints were monitored daily to generate a clinical score. Immunohistology was used to evaluate neutrophil infiltration and TUNEL to assess apoptosis. ELISA was used to measure IL-1β. Results: Treatment with rFc-µTP-L309C, but not HSA, was able to significantly ameliorate the arthritis in the K/BxN mice. Significant neutrophil infiltration into the ankle joint was found, but treatment with rFc-µTP-L309C resulted in significantly less neutrophil infiltration. There was no significant influence of rFc-µTP-L309C on neutrophil death or apoptosis. Less neutrophil infiltration could not be correlated to chemokine-mediated migration. Significantly less IL-1β was measured in mice treated with rFc-µTP-L309C. Conclusion: In the endogenous K/BxN mouse model of rheumatoid arthritis, amelioration can be explained in part by inhibition of neutrophil infiltration into the joints as well as inhibition of IL-1β production. Given the observed inhibitory properties on neutrophils, rFc-µTP-L309C may be a potential therapeutic candidate to treat autoimmune and inflammatory conditions in which neutrophils are the predominant cell type involved in pathogenesis.

Artemisinin-derived artemisitene blocks ROS-mediated NLRP3 inflammasome and alleviates ulcerative colitis

Artemisinins are well-known antimalarial drugs with clinical safety. In addition to antimalarial effects, their anti-inflammatory and immunoregulatory properties have recently attracted much attention in the treatment of inflammatory diseases. However, these artemisinins only have sub-millimolar anti-inflammatory activity in vitro, which may pose a high risk of toxicity in vivo with high doses of artemisinins. Here, we identified another derivative, artemisitene, which can increase the activity of inhibiting the NLRP3 pathway by more than 200-fold through introducing a covalent binding group while retaining the peroxide bridge structure. Mechanistically, artemisitene inhibits the production of ROS (especially mtROS) and prevents the assembly and activation of NLRP3 inflammasome, thereby inhibiting IL-1β production. In addition, it can also block IL-1β secretion mediated by NLRC4 and AIM2 inflammasome and IL-6 production. Furthermore, treatment with artemisitene significantly attenuated inflammatory response in DSS-induced ulcerative colitis. Our work provides a potential artemisinin derivative, which is worthy of further structural optimization based on pharmacokinetic properties as a drug candidate for inflammatory disorders.

Corilagin Restrains NLRP3 Inflammasome Activation and Pyroptosis through the ROS/TXNIP/NLRP3 Pathway to Prevent Inflammation.

Corilagin, a gallotannin, shows excellent antioxidant and anti-inflammatory effects. The NLRP3 inflammasome dysfunction has been implicated in a variety of inflammation diseases. However, it remains unclear how corilagin regulates the NLRP3 inflammasome to relieve gouty arthritis. In this study, bone marrow-derived macrophages (BMDMs) were pretreated with lipopolysaccharide (LPS) and then incubated with NLRP3 inflammasome agonists, such as adenine nucleoside triphosphate (ATP), nigericin, and monosodium urate (MSU) crystals. The MSU crystals were intra-articular injected to induce acute gouty arthritis. Here we showed that corilagin reduced lactate dehydrogenase (LDH) secretion and the proportion of propidium iodide- (PI-)stained cells. Corilagin suppressed the expression of N-terminal of the pyroptosis executive protein gasdermin D (GSDMD-NT). Corilagin restricted caspase-1 p20 and interleukin (IL)-1β release. Meanwhile, corilagin attenuated ASC oligomerization and speck formation. Our findings confirmed that corilagin diminished NLRP3 inflammasome activation and macrophage pyroptosis. We further discovered that corilagin limited the mitochondrial reactive oxygen species (ROS) production and prevented the interaction between TXNIP and NLRP3, but ROS activator imiquimod could antagonize the inhibitory function of corilagin on NLRP3 inflammasome and macrophage pyroptosis. Additionally, corilagin ameliorated MSU crystals induced joint swelling, inhibited IL-1β production, and abated macrophage and neutrophil migration into the joint capsule. Collectively, these results demonstrated that corilagin suppressed the ROS/TXNIP/NLRP3 pathway to repress inflammasome activation and pyroptosis and suggest its potential antioxidative role in alleviating NLRP3-dependent gouty arthritis.

Interactions between the NLRP3-Dependent IL-1β and the Type I Interferon Pathways in Human Plasmacytoid Dendritic Cells.

Generally, a reciprocal antagonistic interaction exists between the antiviral type I interferon (IFN) and the antibacterial nucleotide-binding oligomerization domain (NOD)-like receptor pyrin domain containing 3 (NLRP3)-dependent IL-1β pathways that can significantly shape immune responses. Plasmacytoid dendritic cells (pDCs), as professional type I IFN-producing cells, are the major coordinators of antiviral immunity; however, their NLRP3-dependent IL-1β secretory pathway is poorly studied. Our aim was to determine the functional activity of the IL-1β pathway and its possible interaction with the type I IFN pathway in pDCs. We found that potent nuclear factor-kappa B (NF-κB) inducers promote higher levels of pro-IL-1β during priming compared to those activation signals, which mainly trigger interferon regulatory factor (IRF)-mediated type I IFN production. The generation of cleaved IL-1β requires certain secondary signals in pDCs and IFN-α or type I IFN-inducing viruses inhibit IL-1β production of pDCs, presumably by promoting the expression of various NLRP3 pathway inhibitors. In line with that, we detected significantly lower IL-1β production in pDCs of psoriasis patients with elevated IFN-α levels. Collectively, our results show that the NLRP3-dependent IL-1β secretory pathway is inducible in pDCs; however, it may only prevail under inflammatory conditions, in which the type I IFN pathway is not dominant.

Pycard and BC017158 Candidate Genes of Irm1 Locus Modulate Inflammasome Activation for IL-1β Production.

We identified Pycard and BC017158 genes as putative effectors of the Quantitative Trait locus (QTL) that we mapped at distal chromosome 7 named Irm1 for Inflammatory response modulator 1, controlling acute inflammatory response (AIR) and the production of IL-1β, dependent on the activation of the NLRP3 inflammasome. We obtained the mapping through genome-wide linkage analysis of Single Nucleotide Polymorphisms (SNPs) in a cross between High (AIRmax) and Low (AIRmin) responder mouse lines that we produced by several generations of bidirectional selection for Acute Inflammatory Response. A highly significant linkage signal (LOD score peak of 72) for ex vivo IL-1β production limited a 4 Mbp interval to chromosome 7. Sequencing of the locus region revealed 14 SNPs between “High” and “Low” responders that narrowed the locus to a 420 Kb interval. Variants were detected in non-coding regions of Itgam, Rgs10 and BC017158 genes and at the first exon of Pycard gene, resulting in an E19K substitution in the protein ASC (apoptosis associated speck-like protein containing a CARD) an adaptor molecule in the inflammasome complex. Silencing of BC017158 inhibited IL1-β production by stimulated macrophages and the E19K ASC mutation carried by AIRmin mice impaired the ex vivo IL-1β response and the formation of ASC specks in stimulated cells. IL-1β and ASC specks play major roles in inflammatory reactions and in inflammation-related diseases. Our results delineate a novel genetic factor and a molecular mechanism affecting the acute inflammatory response.

Activation of Canine, Mouse and Human TLR2 and TLR4 by Inactivated Leptospira Vaccine Strains.

Canine Leptospira vaccines contain inactivated strains of pathogenic Leptospira, the causative agents of leptospirosis. For an effective response to vaccination, activation of the innate immune system via pattern recognition receptors such as TLRs is crucial. However, it is not known which TLRs are activated by Leptospira in dogs. To investigate the involvement of canine TLR2, TLR4, and TLR5 in the recognition of Leptospira, we stimulated canine moDC and reporter cells expressing canine TLR2 with either whole-inactivated bacteria or purified LPS of Leptospira strains, representing the serogroups generally used in canine leptospirosis vaccines. Using the endotoxin neutralizing reagent polymyxin B and TLR4 antagonist RS-LPS, we demonstrate that Leptospira LPS and canine TLR4 are involved in IL-1β production as well as in the uptake of inactivated Leptospira in canine moDC. Furthermore, polymyxin B only partially inhibited IL-1β production induced by inactivated Leptospira, suggesting that next to TLR4, also other TLRs may be involved. The observed activation of canine TLR2-expressing reporter cells by inactivated Leptospira strains indicates that TLR2 could be one of these TLRs. Next, we analyzed TLR2 and TLR4 activating capabilities by the same Leptospira strains using human and mouse TLR-expressing reporter cells. Inactivated Leptospira and leptospiral LPS activated not only mouse, but also human TLR4 and this activation was shown to be LPS dependent in both cases. Additionally, inactivated Leptospira activated mouse and human TLR2-expressing reporter cell lines. In our study, we could not identify significant species differences in the recognition of Leptospira by TLR2 and TLR4 between dog, human and mouse. Lastly, we show that these inactivated Leptospira strains are recognized by both mouse and human TLR5 reporter cells only after exposure to additional heat-treatment. Unfortunately, we were not able to confirm this in the canine system. Our data show that TLR2 and TLR4 are involved in the recognition of Leptospira strains used in the production of canine Leptospira vaccines. This study contributes to the understanding of Leptospira-induced innate immune responses in dogs, humans, and mice. Future studies are needed to further explore the role of canine TLR2, TLR4 and TLR5 in the induction of vaccine-mediated immunity against Leptospira.

Convallatoxin inhibits IL-1β production by suppressing zinc finger protein 91 (ZFP91)-mediated pro-IL-1β ubiquitination and caspase-8 inflammasome activity.

ZFP91 positively regulates IL-1β production in macrophages and may be a potential therapeutic target to treat inflammatory-related diseases. We investigated whether this process is modulated by convallatoxin, which is a cardiac glycoside isolated from the traditional Chinese medicinal plant Adonis amurensis Regel et Radde. In vitro, the mechanisms by which convallatoxin inhibits ZFP91-regulated IL-1β expression were investigated using molecular docking, western blotting, RT-PCR, ELISA, immunofluorescence and immunoprecipitation assays.In vivo, mice liver injury was induced by an intraperitoneal injection of D-GalN and LPS, colitis was induced by oral administration of dextran sulfate sodium (DSS) in drinking water and peritonitis was induced by an intraperitoneal injection of alum. We confirmed that convallatoxin inhibited the release of IL-1β by down-regulating ZFP91. Importantly, we found that convallatoxin significantly reduced K63-linked polyubiquitination of pro-IL-1β regulated by ZFP91 and decreased the efficacy of pro-IL-1β cleavage. Moreover, convallatoxin suppressed ZFP91-mediated activation of the non-canonical cysteine-requiring aspartate protease-8 (caspase-8) inflammasome and MAPK signalling pathways in macrophages. Furthermore, we showed that ZFP91 promoted the assembly of the caspase-8 inflammasome complex, whereas convallatoxin treatment reversed this result. Mice in vivo studies further demonstrated that convallatoxin ameliorated D-GalN/LPS-induced liver injury, DSS-induced colitis and alum-induced peritonitis by down-regulating ZFP91. We show for the first time that convallatoxin-mediated inhibition of ZFP91 is an important regulatory event that prevents inappropriate inflammatory responses to maintain immune homeostasis. This mechanism provides new insight for the development of convallatoxin as a novel anti-inflammatory drug targeting ZFP91. This article is part of a themed issue on Inflammation, Repair and Ageing. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.9/issuetoc.

Benzyl Isothiocyanate Attenuates Inflammasome Activation in Pseudomonas aeruginosa LPS-Stimulated THP-1 Cells and Exerts Regulation through the MAPKs/NF-κB Pathway.

Inflammasomes are a group of intracellular multiprotein platforms that play important roles in immune systems. Benzyl isothiocyanate (BITC) is a constituent of cruciferous plants and has been confirmed to exhibit various biological activities. The modulatory effects of BITC on inflammasome-mediated interleukin (IL)-1β expression and its regulatory mechanisms in Pseudomonas aeruginosa (P. aeruginosa) LPS/ATP-stimulated THP-1 cells was investigated. Monocytic THP-1 cells were treated with phorbol myristate acetate (PMA) to induce differentiation into macrophages. Enzyme-linked immunosorbent assays (ELISA) were performed to measure the levels of IL-1β produced in P. aeruginosa LPS/ATP-exposed THP-1 cells. Western blotting was performed to examine the BITC modulatory mechanisms in inflammasome-mediated signaling pathways. BITC inhibited IL-1β production in P. aeruginosa LPS/ATP-induced THP-1 cells. BITC also inhibited activation of leucine-rich repeat protein-3 (NLRP3) and caspase-1 in P. aeruginosa LPS/ATP-induced THP-1 cells. Furthermore, we show that mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) activation in P. aeruginosa LPS was attenuated by BITC. These BITC-mediated modulatory effects on IL-1β production may have therapeutic potential for inflammasome-mediated disorders such as a nasal polyp.

Paeonol reduces IL-β production by inhibiting the activation of nucleotide oligomerization domain-like receptor protein-3 inflammasome and nuclear factor-κB in macrophages.

Interleukin-1β, a key cytokine in gouty inflammation, is precisely regulated by the NLRP3 inflammasome and NF-κB. Our previous study demonstrated that paeonol suppressed IL-1β production in rats with monosodium urate (MSU)-induced arthritis. Whether NLRP3 inflammasome or NF-κB is responsible for the anti-inflammatory effect of paeonol remains unclear. In this study, J774A.1 cells induced by lipopolysaccharide (LPS) plus MSU, was used to investigate the effect of paeonol on NLRP3 inflammasome activation, and J774A.1 cells induced by LPS alone were used to investigate the effect of paeonol on NF-κB activation. In J774A.1 cells induced by LPS plus MSU, paeonol decreased the levels of IL-1β and caspase-1 and reduced the MSU-induced interaction of pro-caspase-1 and apoptosis-associated speck-like protein containing caspase recruitment domain (ASC), but did not affect the levels of pro-IL-1β and pro-caspase-1. In J774A.1 cells induced by LPS alone, paeonol reduced the levels of IL-1β, NLRP3, p-IKK, p-IκBα, and p-p65, but did not affect ASC levels. Paeonol also promoted the content of IκBα and retained more p65 in the cytoplasm. Furthermore, paeonol reduced the DNA-binding activity of p65 and lowered the levels of p-JNK, p-ERK, and p-p38. These results suggest that paeonol inhibits IL-1β production by inhibiting the activation of NLRP3 inflammasome, NF-κB, and MAPK signaling pathways.

Effects of Orally Administered Cannabidiol on Neuroinflammation and Intestinal Inflammation in the Attenuation of Experimental Autoimmune Encephalomyelitis.

Cannabidiol (CBD) is a bioactive compound isolated from Cannabis plants that has garnered attention within the medical community due to its potent anti-inflammatory properties. To better understand how CBD limits excessive neuroinflammation we administered CBD via oral gavage (20 mg/kg) in a murine model of multiple sclerosis (MS) known as experimental autoimmune encephalomyelitis (EAE). Using single cell RNA sequencing (scRNA Seq) and array-based transcriptomics we were able to delineate how CBD limits excessive inflammation within the central nervous system (CNS) as well as within the intestinal lining in EAE. In-depth scRNA Seq analysis of CNS tissue demonstrated that CBD treatment resulted in a significant reduction in CXCL9, CXCL10 and IL-1β expression within the CNS, leading to inhibited infiltration of inflammatory macrophages. CBD inhibited IL-1β production independent of the classical cannabinoid receptors, CB1 and CB2. CBD treatment also led to induction of Myeloid-derived Suppressor Cells (MDSCs) both in the CNS and periphery. Interestingly, CBD treatment of EAE mice revealed significant suppression of inflammation in the gastrointestinal (GI) tract. The intestinal epithelial cells (IECs) of CBD treated mice demonstrated a transcriptional inhibition of a family of pyroptosis initiators that drive localized inflammation known as gasdermins (GSDMs). Further investigation into the GI tract via 16s sequencing of cecal and fecal contents demonstrated that oral administration of CBD resulted in no significant changes in the intestinal microbiota composition. These findings demonstrate the beneficial effect of CBD treatment on autoimmune neuroinflammation by ablating expression of pro-inflammatory chemoattractants, regulating inflammatory macrophage activity, promoting MDSC expansion, and limiting the systemic low-grade inflammation in the GI tract, culminating in the attenuation of EAE.

Macrophages regulates the transition of pericyte to peritoneal fibrosis through the GSDMD/IL-1β axis

BackgroundEnd stage renal disease (ESRD) has caused public health problem with high prevalence worldwide. Peritoneum from peritoneal dialysis patients with ESRD can induce pathological changes of the peritoneum, including fibrosis. The trans-differentiation of pericytes has been found to be closely associated with inflammatory diseases, such as organ fibrosis. However, the function of macrophages in regulating the transition of pericyte to peritoneal fibrosis is unclear. MethodsHistological examination was conducted using Hematoxylin and eosin (HE) staining and Masson’s trichrome staining. The protein levels were determined via western blot. Enzyme-linked immunosorbent assay (ELISA) was used to examine IL-1β concentrations. Gasdermin D (GSDMD) was knocked out in mice by Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-Associated 9 (CRISPR-Cas9). ResultsMice receiving dextrose peritoneal dialysate displayed mesothelial cell monolayer loss and thickness of submesothelial compact zone increase. Moreover, dextrose peritoneal dialysate treatment up-regulated GSDMD expression. GSDMD knockdown inhibited IL-1β production in macrophages. Further, pericytes were treated with cultural supernatant from macrophages. We found that GSDMD knockdown suppressed fibrosis and vascular endothelial growth factor (VEGF)/phosphoinositide 3-kinase (PI3K) pathway in pericytes. In addition, GSDMD were knocked out in mice using CRISPR/Cas9. The histological examinations revealed that GSDMD−/− alleviated the damage of peritoneal tissue and thickness of submesothelial compact zone. GSDMD−/− attenuated interleukin-1beta (IL-1β) level and peritoneal fibrosis induced by dextrose peritoneal dialysate treatment in pericytes in vivo. ConclusionThese results demonstrated that macrophages can regulate the transition of pericyte to peritoneal fibrosis via the GSDMD/IL-1β axis, which provides a new therapeutic target.

The Specific NLRP3 Antagonist IFM-514 Decreases Fibrosis and Inflammation in Experimental Murine Non-Alcoholic Steatohepatitis.

Background and Aims: Activation of the inflammasome NLRP3 (NOD-, LRR- and pyrin domain containing 3) contributes to the development of non-alcoholic fatty liver disease (NAFLD) and progression to non-alcoholic steatohepatitis (NASH). Therefore, this study explored the therapeutic effects of a novel and selective NLRP3 antagonist in a murine dietary model of NASH.Methods: Groups of 12-week-old ApoE -/- mice were fed ad lib for 7 weeks with a methionine/choline deficient (MCD) and western diet (WD). After 3 weeks of diet-induced injury, mice were injected i. p. with the NLRP3 antagonist IFM-514 (100 mg/kg body weight) or vehicle (0.5% carmellose) every day, 5 days/week for a further 4 weeks. Several markers of inflammation, fibrosis and steatosis were evaluated. Whole transcriptome sequencing and panel RNA expression analysis (NanoString) were performed.Results: IFM-514 inhibited IL-1β production in mice challenged with 20 mg/kg lipopolysaccharide, and in mouse and human inflammatory cells in vitro. IFM-514 inhibited hepatic inflammation in the in vivo non-alcoholic steatohepatitis model assessed by H&E staining and in the hepatic gene expression of inflammasome-related proinflammatory cytokines. This effect was associated with significant reduction in caspase-1 activation. Similarly, IFM-514 was efficacious in vivo in MDC-fed ApoE -/- mice, markedly reducing portal pressure, Sirius red staining and 4-hydroxyproline content compared to vehicle-treated mice. Moreover, IFM-514 significantly reduced hepatic steatosis in MCD-fed ApoE -/- mice, as evidenced by NAFLD scores, oil red O staining, hepatic triglycerides and gene expression. In WD treated animals, similar trends in inflammation and fibrosis were observed, although not sufficient IFM-514 levels were reached.Conclusion: Overall, IFM-514 reduced liver inflammation and fibrosis, with mild effects on liver steatosis in experimental murine NASH. Blocking of NLRP3 may be an attractive therapeutic approach for NASH patients.

Protective Effect of Piplartine against LPS-Induced Sepsis through Attenuating the MAPKs/NF-κB Signaling Pathway and NLRP3 Inflammasome Activation.

Piplartine (or Piperlongumine) is a natural alkaloid isolated from Piper longum L., which has been proposed to exhibit various biological properties such as anti-inflammatory effects; however, the effect of piplartine on sepsis has not been examined. This study was performed to examine the anti-inflammatory activities of piplartine in vitro, ex vivo and in vivo using murine J774A.1 macrophage cell line, peritoneal macrophages, bone marrow-derived macrophages and an animal sepsis model. The results demonstrated that piplartine suppresses iNOS and COX-2 expression, reduces PGE2, TNF-α and IL-6 production, decreases the phosphorylation of MAPKs and NF-κB and attenuates NF-κB activity by LPS-activated macrophages. Piplartine also inhibits IL-1β production and suppresses NLRP3 inflammasome activation by LPS/ATP- and LPS/nigericin-activated macrophages. Moreover, piplartine reduces the production of nitric oxide (NO) and TNF-α, IL-6 and IL-1β, decreases LPS-induced tissue damage, attenuates infiltration of inflammatory cells and enhances the survival rate. Collectively, these results demonstrate piplartine exhibits anti-inflammatory activities in LPS-induced inflammation and sepsis and suggest that piplartine might have benefits for sepsis treatment.

Binding of Macrophage Receptor MARCO, LDL, and LDLR to Disease-Associated Crystalline Structures.

Endogenous and exogenous crystalline structures are involved in various pathologies and diseases in humans by inducing sterile inflammation, mechanical stress, or obstruction of excretory organs. The best studied of these diseases is gout, in which crystallization of uric acid in the form of monosodium urate (MSU) mainly in synovial fluid of the joints leads to sterile inflammation. Though some of these diseases have been described for centuries, little is known about if and how the immune system recognizes the associated crystals. Thus, in this study we aimed at identifying possible recognition molecules of MSU using liquid chromatography-mass spectrometry (LC-MS) analysis of MSU-binding serum proteins. Among the strongest binding proteins, we unexpectedly found two transmembrane receptors, namely macrophage receptor with collagenous structure (MARCO) and low-density lipoprotein (LDL) receptor (LDLR). We show that recombinant versions of both human and mouse MARCO directly bind to unopsonized MSU and several other disease-associated crystals. Recombinant LDLR binds many types of crystals mainly when opsonized with serum proteins. We show that this interaction is predominantly mediated by LDL, which we found to bind to all crystalline structures tested except for cholesterol crystals. However, murine macrophages lacking LDLR expression do neither show altered phagocytosis nor interleukin-1β (IL-1β) production in response to opsonized crystals. Binding of LDL to MSU has previously been shown to inhibit the production of reactive oxygen species (ROS) by human neutrophils. We extend these findings and show that LDL inhibits neutrophil ROS production in response to most crystals tested, even cholesterol crystals. The inhibition of neutrophil ROS production only partly correlated with the inhibition of IL-1β production by peripheral blood mononuclear cells (PBMCs): LDL inhibited IL-1β production in response to large MSU crystals, but not small MSU or silica crystals. This may suggest distinct upstream signals for IL-1β production depending on the size or the shape of the crystals. Together, we identify MARCO and LDLR as potential crystal recognition receptors, and show that LDL binding to diverse disease-associated crystalline structures has variable effects on crystal-induced innate immune cell activation.

The NLRP3 inhibitor MCC950 inhibits IL-1β production in PBMC from 19 patients with Cryopyrin-Associated Periodic Syndrome and in 2 patients with Schnitzler’s Syndrome

Background: The cryopyrin-associated periodic syndromes (CAPS) are a group of inherited disorders associated with systemic auto-inflammation. CAPS result from gain-of-function mutations in NLRP3, which result in formation of an intracellular protein complex known as the NLRP3 inflammasome. This leads to overproduction of IL-1β and other pro-inflammatory signals, resulting in inflammatory symptoms. Treatments for NLRP3-related diseases are biologic agents that directly target IL-1β. We sought to determine if the orally available small molecule NLRP3 inhibitor MCC950 could inhibit IL-1β ex vivo in a cohort of patients with autoinflammatory disease. Methods: Patients were recruited to donate blood, from which PBMCs were isolated and assayed in the presence of MCC950 to determine inhibitory efficacy. Results: We found that apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and mature IL-1β was higher in ex vivo PBMCs from CAPS patients than healthy donors. MCC950 inhibited production of mature IL-1β in PBMC from CAPS patients with a range of mutations and blocked NLRP3 activity in an in vitro mutation reconstitution assay. Similar results were observed with PBMC from two patients with Schnitzler’s Syndrome, another auto-inflammatory disease. Conclusions: The NLRP3 inflammasome inhibitor MCC950 blocked constitutive activation of NLRP3 observed in the PBMCs of CAPS patients. This study highlights the potential utility of NLRP3 inhibition by a small molecule for rare autoinflammatory diseases that are driven by NLRP3.

Interleukin-1β in hypothalamic paraventricular nucleus mediates excitatory renal reflex.

Chemical stimulation of kidney causes sympathetic activation and pressor responses in rats. The excitatory renal reflex (ERR) is mediated by angiotensin type 1 receptor (AT1R) and superoxide anions in hypothalamic paraventricular nucleus (PVN). The aim of this study is to determine whether interleukin-1β (IL-1β) in the PVN mediates the ERR, and whether the IL-1β production in the PVN is dependent on the AT1R-superoxide anion signaling. Experiments were performed in adult rats under anesthesia. The ERR was induced by renal infusion of capsaicin, and evaluated by the responses of the contralateral renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP). Inhibition of IL-1β production with MCC950 in the PVN dose-dependently inhibited the capsaicin-induced ERR and sympathetic activation. The PVN microinjection of IL-1 receptor antagonist IL-1Ra or specific IL-1β antibody abolished the capsaicin-induced ERR, while IL-1β enhanced the ERR. Renal infusion of capsaicin promoted p65-NFκB phosphorylation and IL-1β production in the PVN, which were prevented by PVN microinjection of NADPH oxidase inhibitor apocynin or the superoxide anion scavenger tempol. The PVN microinjection of NFκB inhibitor BMS-345541 abolished the capsaicin induced-ERR and IL-1β production, but not the NADPH oxidase activation and superoxide anion production. Furthermore, capsaicin-induced p65-NFκB phosphorylation and IL-1β production in the PVN were prevented by AT1R antagonist losartan, or angiotensin converting enzyme inhibitor captopril. These results indicate that capsaicin-induced ERR and sympathetic activation are mediated by IL-1β in the PVN. The IL-1β production in the PVN is dependent on the AT1R-mediated superoxide anion generation and NFκB activation.