Beta Secretion Research Articles

EPA and DHA inhibit LDL-induced upregulation of human adipose tissue NLRP3 inflammasome/IL-1β pathway and its association with diabetes risk factors

Elevated numbers of atherogenic lipoproteins (apoB) predict the incidence of type 2 diabetes (T2D). We reported that this may be mediated via the activation of the NLRP3 inflammasome, as low-density lipoproteins (LDL) induce interleukin-1 beta (IL-1β) secretion from human white adipose tissue (WAT) and macrophages. However, mitigating nutritional approaches remained unknown. We tested whether omega-3 eicosapentaenoic and docosahexaenoic acids (EPA and DHA) treat LDL-induced upregulation of WAT IL-1β-secretion and its relation to T2D risk factors. Twelve-week intervention with EPA and DHA (2.7 g/day, Webber Naturals) abolished baseline group-differences in WAT IL-1β-secretion between subjects with high-apoB (N = 17) and low-apoB (N = 16) separated around median plasma apoB. Post-intervention LDL failed to trigger IL-1β-secretion and inhibited it in lipopolysaccharide-stimulated WAT. Omega-3 supplementation also improved β-cell function and postprandial fat metabolism in association with higher blood EPA and mostly DHA. It also blunted the association of WAT NLRP3 and IL1B expression and IL-1β-secretion with multiple cardiometabolic risk factors including adiposity. Ex vivo, EPA and DHA inhibited WAT IL-1β-secretion in a dose-dependent manner. In conclusion, EPA and DHA treat LDL-induced upregulation of WAT NLRP3 inflammasome/IL-1β pathway and related T2D risk factors. This may aid in the prevention of T2D and related morbidities in subjects with high-apoB.Clinical Trail Registration ClinicalTrials.gov (NCT04496154): Omega-3 to Reduce Diabetes Risk in Subjects with High Number of Particles That Carry “Bad Cholesterol” in the Blood – Full Text View - ClinicalTrials.gov.

Aiouea padiformis extract exhibits anti-inflammatory effects by inhibiting the ATPase activity of NLRP3

Inflammation is implicated as a cause in many diseases. Most of the anti-inflammatory agents in use are synthetic and there is an unmet need for natural substance-derived anti-inflammatory agents with minimal side effects. Aiouea padiformis belongs to the Lauraceae family and is primarily found in tropical regions. While some members of the Aiouea genus are known to possess anti-inflammatory properties, the anti-inflammatory properties of Aiouea padiformis extract (AP) have not been investigated. In this study, we aimed to examine the anti-inflammatory function of AP through the NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome and elucidate the underlying mechanisms. Treatment with AP inhibited the secretion of interleukin-1 beta (IL-1β) mediated by NLRP3 inflammasome in J774A.1 and THP-1 cells without affecting the viability. In addition, AP treatment did not influence NF-κB signaling, potassium efflux, or intracellular reactive oxygen species (ROS) production—all of which are associated with NLRP3 inflammasome activation. However, intriguingly, AP treatment significantly reduced the ATPase activity of NLRP3, leading to the inhibition of ASC oligomerization and speck formation. Consistent with cellular experiments, the anti-inflammatory property of AP in vivo was also evaluated using an LPS-induced inflammation model in zebrafish, demonstrating that AP hinders NLRP3 inflammasome activation.

The Prolonged Activation of the p65 Subunit of the NF-Kappa-B Nuclear Factor Sustains the Persistent Effect of Advanced Glycation End Products on Inflammatory Sensitization in Macrophages.

Advanced glycation end products (AGEs) prime macrophages for lipopolysaccharide (LPS)-induced inflammation. We investigated the persistence of cellular AGE-sensitization to LPS, considering the nuclear content of p50 and p65 nuclear factor kappa B (NFKB) subunits and the expression of inflammatory genes. Macrophages treated with control (C) or AGE-albumin were rested for varying intervals in medium alone before being incubated with LPS. Comparisons were made using one-way ANOVA or Student t-test (n = 6). AGE-albumin primed macrophages for increased responsiveness to LPS, resulting in elevated levels of TNF, IL-6, and IL-1beta (1.5%, 9.4%, and 5.6%, respectively), compared to C-albumin. TNF, IL-6, and IL-1 beta secretion persisted for up to 24 h even after the removal of AGE-albumin (area under the curve greater by 1.6, 16, and 5.2 times, respectively). The expressions of Il6 and RelA were higher 8 h after albumin removal, and Il6 and Abca1 were higher 24 h after albumin removal. The nuclear content of p50 remained similar, but p65 showed a sustained increase (2.9 times) for up to 24 h in AGE-albumin-treated cells. The prolonged activation of the p65 subunit of NFKB contributes to the persistent effect of AGEs on macrophage inflammatory priming, which could be targeted for therapies to prevent complications based on the AGE-RAGE-NFKB axis.

Cell membrane-anchored and tumor-targeted IL-12 T-cell therapy destroys cancer-associated fibroblasts and disrupts extracellular matrix in heterogenous osteosarcoma xenograft models

BackgroundThe extracellular matrix (ECM) and cancer-associated fibroblasts (CAFs) play major roles in tumor progression, metastasis, and the poor response of many solid tumors to immunotherapy. CAF-targeted chimeric antigen receptor-T cell...

“Kupffer Cell Teleportation” Strategy for Liver Fibrosis Alleviation Based on Hybrid Polymer‐ Bimetallic Sequential Delivery System

AbstractAlthough direct hepatic stellate cell (HSC) inhibition is considered a promising strategy for alleviating liver fibrosis, traditional therapies struggle with off‐target effects due to limited penetration of liver sinusoidal endothelial cell barriers. This study reports on the development of a “Kupffer cell (KC) teleportation” strategy to treat liver fibrosis by fabricating a hybrid polymer‐bimetallic sequential delivery system, CF@BPPM (baicalin prodrug polymer [BP]/mannose derivative polymer [PM] encapsulated copper [Cu]‐iron [Fe] ultrasmall nanoparticles [CFs]). The nanosystems exhibit KCs targeting and acidic response sequential release properties by a hybrid polymer, which first releases CFs in lysosome after being internalized by KCs through a PM targeting effect. The exposed CFs efficiently scavenge reactive oxygen species in KCs and subsequently degrade Cu2+/ Fe2+ to upregulate the heme oxygenase expression for suppressing interleukin 1β, interleukin 6, tumor necrosis factor‐alpha, and platelet‐derived growth factors. Second, baicalin, released from BP in lysosome, further inhibits transforming growth factor beta secretion. These results suggest that CF@BPPM inhibits the inclusive cytokine secretion of KCs, which play a vital conductive role in liver fibrosis progression. Subsequently, CF@BPPM achieves protection against HSCs activation, migration, and collagen production, enriching anti‐liver fibrosis therapeutic methods by exploiting messaging between KCs and HSCs.

The role of the biofilm of Porphyromonas gingivalis in driving amyloid beta secretion and aggregation

AbstractBackgroundOver the past five years, the hypothesis linking periodontitis to Alzheimer’s disease (AD) has gained significant traction even though little is known about the underlying mechanisms driving this association. Periodontitis is caused by the dysbiosis of the dental biofilm, an extra‐cellular matrix produced by bacteria, which is mostly caused by the Gram‐negative bacterium Porphyromonas gingivalis. P. gingivalis has been found to infect the brain of AD patients and drive amyloid beta (Aβ) production while biofilm components can be identified interwoven with Aβ plaques1,2. We therefore hypothesize that P. gingivalis drives Aβ production in neural cells which co‐aggregates with bacterial biofilm to expedite plaque formation and downstream neuroinflammation.MethodTo assess if the biofilm of co‐aggregation of Aβ and the matrix was then analyzed by fluorescence microscopy, atomic force microscopy (AFM) and thioflavin T (ThT) fluorescence assays. To examine whether P. gingivalis stimulates Aβ production, we infected BE(2)‐M17 human neuroblastomas at a mean of infection of 1:100 before collecting cells and supernatant proteins to observe the cell response through protein and transcription assays.ResultsWhen co‐cultured, P. gingivalis and Aβ1‐42 co‐aggregated in plaque‐like structures, Interestingly, colocalization analysis revealed that Aβ1‐42 particularly associate with the extracellular component of biofilm (Figure 1). ThT assays and AFM imaging demonstrated faster aggregation of Aβ1‐42 when co‐incubated with fragments of P. gingivalis biofilm for 8 h, which suggests a significant cross‐seeding phenomenon between the two components (Figure 2). Furthermore, infection of human neuroblastomas resulted in a significant increase in Aβ production that could be detected by western blot, thus offering a workable model to study the link between AD and periodontitis (Figure 3).ConclusionOur project offers significant insights into the relationship between periodontitis and AD, allowing us to understand how P. gingivalis triggers Aβ production and how its biofilm drives plaque aggregation. This research greatly contributes to a better understanding of external AD risk factors and how to mediate their impacts on neurodegeneration. 1Dominy et al. Science Advances (2019) 2Mirzaei et al. Microbial Pathogenesis (2020)

Hepatitis C Virus Nonstructural Protein 3 Increases Secretion of Interleukin-1beta in HEK293T Cells with Reconstructed NLRP3 Inflammasome

Infections caused by Flaviviridae pose a threat in the modern world. The pathology of diseases arising from these infections is largely determined by the development of systemic inflammation. The cytokines interleukin-1 beta and interleukin-18 play a key role in triggering inflammation. Their secretion from cells, in its turn, is induced upon activation of inflammasomes. Activation of NLRP3 (NLR family pyrin domain-containing 3) inflammasomes was detected in the cells infected with Flaviviridae. Some nonstructural proteins of these viruses have been shown to be able to activate or inhibit the NLRP3 inflammasome, in particular, through interaction with its components. In this study, the functional NLRP3 inflammasome was reconstructed in human HEK293T cells and the effect of some nonstructural proteins of individual Flaviviridae viruses on it was studied. This model did not reveal any impact of nonstructural proteins NS1 of West Nile virus, NS3 of hepatitis C virus, NS5 of tick-borne encephalitis virus on the inflammasome components content. At the same time, in the presence of the NS1 of the West Nile virus and NS5 of the tick-borne encephalitis virus, the level of secretion of interleukin-1 beta did not change, whereas in the presence of the NS3 protein of the hepatitis C virus, it increased by 1.5 times. Thus, NS3 can be considered as one of the factors of NLRP3 inflammasome activation and inflammatory pathogenesis in chronic hepatitis C virus infection.

CXCL5 promotes lipotoxicity of hepatocytes through upregulating NLRP3/Caspase-1/IL-1β signaling in Kupffer cells and exacerbates nonalcoholic steatohepatitis in mice

Immune-inflammatory responses play a key role in the development of nonalcoholic steatohepatitis (NASH). Previous studies have demonstrated that CXC motif chemokine ligand 5 (CXCL5) correlates positively with obesity and type 2 diabetes. This study is to explore the functional role of CXCL5 in the pathogenesis of NASH. To establish a NASH model, mice were fed with methionine-and choline-deficient high-fat diet for 6 weeks and anti-CXCL5 mAb was injected during the same period. An in vitro NASH model was established by treating palmitic acid (PA), using a trans-well co-culture system of mouse primary hepatocytes and Kupffer cells (KCs), and recombinant mouse (rm) CXCL5 was treated after PA administration. Our data showed that hepatic CXCL5 levels were highly expressed in the NASH mouse model. CXCL5 neutralization significantly alleviated the severity of NASH livers, demonstrated by pathological analysis, decreased biochemicals, and inflammation. Besides, neutralizing CXCL5 reduced lipid accumulation, cell death, and fibrosis in injured livers. In vitro, rmCXCL5 could not affect the activation of hepatic stellate cells. Also, rmCXCL5 exacerbated PA-induced hepatotoxicity and lipid deposition in hepatocytes co-cultured with KCs rather than in single-cultured hepatocytes. Mechanistically, rmCXCL5 not only promoted NOD-like receptor pyrin domain-containing protein 3 (NLRP3) expression, Cleaved caspase-1 expression, and interleukin 1 beta (IL-1β) secretion in single-cultured and co-cultured KCs but also increased lipid deposition in co-cultured hepatocytes. In addition, MCC950, an inhibitor of NLRP3, almost abolished the effects of rmCXCL5 on PA-treated co-culture system. Therefore, CXCL5 could exacerbate NASH by promoting lipotoxicity of hepatocytes via upregulating NLRP3/Caspase-1/IL-1β signaling in KCs.

Pyroptosis -related potential diagnostic biomarkers in steroid-induced osteonecrosis of the femoral head

PurposeSteroid-induced necrosis of the femoral head (SONFH) is a refractory orthopedic hip disease occurring in young and middle-aged people, with glucocorticoids being the most common cause. Previous experimental studies have shown that cell pyroptosis may be involved in the pathological process of SONFH, but its pathogenesis in SONFH is still unclear. This study aims to screen and validate potential pyroptosis-related genes in SONFH diagnosis by bioinformatics analysis to further elucidate the mechanism of pyroptosis in SONFH.MethodsThere were 33 pyroptosis-related genes obtained from the prior reviews. The mRNA expression was downloaded from GSE123568 dataset in the Gene Expression Omnibus (GEO) database, including 10 non-SONFH (following steroid administration) samples and 30 SONFH samples. The pyroptosis-related differentially expressed genes involved in SONFH were identified with “affy” and “limma” R package by intersecting the GSE123568 dataset with pyroptosis genes. In addition, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of the pyroptosis-related differentially expressed genes involved in SONFH were conducted by “clusterProfiler” R package and visualized by “GOplot” R package. Then, the correlations between the expression levels of the pyroptosis-related differentially expressed genes involved in SONFH were confirmed with “corrplot” R package. Moreover, the protein–protein interaction (PPI) network was analysed by using GeneMANIA database. Next, The ROC curve of pyroptosis-related differentially expressed genes were analyzed by “pROC” R package.ResultsA total of 10 pyroptosis-related differentially expressed genes were identified between the peripheral blood samples of SONFH patients and non-SONFH patients based on the defined criteria, including 20 upregulated genes and 10 downregulated genes. The GO and KEGG pathway enrichment analyses revealed that these 10 pyroptosis-related differentially expressed genes involved in SONFH were particularly enriched in cysteine-type endopeptidase activity involved in apoptotic process, positive regulation of interleukin-1 beta secretion and NOD-like receptor signaling pathway. Correlation analysis revealed significant correlations among the 10 differentially expressed pyroptosis-related genes involved in SONFH. The PPI results demonstrated that the 10 pyroptosis-related differentially expressed genes interacted with each other. Compared to non-SONFH samples, these pyroptosis-related differentially expressed genes had good predictive diagnostic efficacy (AUC = 1.000, CI = 1.000–1.000) in the SONFH samples, and NLRP1 had the highest diagnostic value (AUC: 0.953) in the SONFH samples.ConclusionsThere were 10 potential pyroptosis-related differentially expressed genes involved in SONFH were identified via bioinformatics analysis, which might serve as potential diagnostic biomarkers because they regulated pyroptosis. These results expand the understanding of SONFH associated with pyroptosis and provide new insights to further explore the mechanism of action and diagnosis of pyroptosis associated in SONFH.

1570-P: Myeloid-Specific Inactivation of Basp1 Protects Mice from Diet-Induced NASH through Attenuating NLRP3 Inflammasome Activation

Nonalcoholic steatohepatitis (NASH) is characterized by persistent liver injury, inflammation, and fibrosis. Myeloid cells, including macrophages and dendritic cells, play an integral role in host defense, tissue homeostasis, and disease progression. Despite this, how myeloid cells contribute to NASH pathogenesis remains obscure. We previously identified NASH-associated macrophages (NAMs) as a unique population of monocyte-derived macrophages in NASH liver. We observed that hepatic NAMs exhibited abundant expression of Brain acid-soluble protein 1 (Basp1), which was markedly induced in the liver upon diet-induced NASH in mice. We generated myeloid-specific Basp1 knockout (Mye-Basp1 KO) mice to determine its role in NASH pathogenesis. Compared to control, Mye-Basp1 KO mice displayed significantly improved liver injury and fibrosis following NASH diet feeding, as shown by lower plasma ALT/AST levels and reduced Sirius Red staining on liver sections, respectively. Hepatocytes from Mye-Basp1 KO mice contained smaller lipid droplets without altering the total liver triglyceride content. Mechanistically, bone marrow-derived macrophages (BMDMs) lacking Basp1 exhibited diminished response to pro-inflammatory stimuli. Importantly, Basp1-deficient BMDMs displayed impaired activation of the NLRP3 inflammasome and secretion of interleukin-1 beta. Together, we propose that Basp1 is a novel regulator of inflammatory signaling in myeloid cells that plays a critical role in NASH pathogenesis. Disclosure Z.Meng: None. L.Zhou: None. S.Hong: None. T.Liu: None. X.Qiu: None. Z.Chen: None. S.Li: None. K.Inoki: None. J.Lin: None. Funding National Institute of Diabetes and Digestive and Kidney Diseases (DK102456)

SARS-CoV-2 omicron sub-lineages differentially modulate interferon response in human lung epithelial cells

Although most of the attention was focused on the characterization of changes in the Spike protein among variants of SARS-CoV-2 virus, mutations outside the Spike region are likely to contribute to virus pathogenesis, virus adaptation and escape to the immune system. Phylogenetic analysis of SARS-CoV-2 Omicron strains reveals that several virus sub-lineages could be distinguished, from BA.1 up to BA.5. Regarding BA.1, BA.2 and BA.5, several mutations concern viral proteins with antagonistic activity to the innate immune system, such as NSP1 (S135R), which is involved in mRNAs translation, exhibiting a general shutdown in cellular protein synthesis. Additionally, mutations and/or deletions in the ORF6 protein (D61L) and in the nucleoprotein N (P13L, D31-33ERS, P151S, R203K, G204R and S413R) have been reported, although the impact of such mutations on protein function has not been further studied. The aim of this study was to better investigate the innate immunity modulation by different Omicron sub-lineages, in the attempt to identify viral proteins that may affect virus fitness and pathogenicity. Our data demonstrated that, in agreement with a reduced Omicron replication in Calu-3 human lung epithelial cells compared to the Wuhan-1 strain, a lower secretion of interferon beta (IFN-β) from cells was observed in all sub-lineages, except for BA.2. This evidence might be correlated with the presence of a mutation within the ORF6 protein (D61L), which is strikingly associated to the antagonistic function of the viral protein, since additional mutations in viral proteins acting as interferon antagonist were not detected or did not show significant influence. Indeed, the recombinant mutated ORF6 protein failed to inhibit IFN-β production in vitro. Furthermore, we found an induction of IFN-β transcription in BA.1 infected cells, that was not correlated with the cytokine release at 72 h post-infection, suggesting that post-transcriptional events can be involved in controlling the innate immunity.

Multiomics Analyses Identify Proline Endopeptidase-Like Protein As a Key Regulator of Protein Trafficking, a Pathway Underlying Alzheimer's Disease Pathogenesis.

Current treatments for Alzheimer's disease (AD) help reduce symptoms for a limited time but do not treat the underlying pathology. To identify potential therapeutic targets for AD, an integrative network analysis was previously carried out using 364 human postmortem control, mild cognitive impairment, and AD brains. This analysis identified proline endopeptidase-like protein (PREPL), an understudied protein, as a downregulated protein in late-onset AD patients. In this study we investigate the role of PREPL. Analyses of data from human postmortem samples and PREPL knockdown (KD) cells suggest that PREPL expression modulates pathways associated with protein trafficking, synaptic activities, and lipid metabolism. Furthermore, PREPL KD impairs cell proliferation and modulates the structure of vesicles, levels of neuropeptide-processing enzymes, and secretion of neuropeptides. In addition, decrease in PREPL levels leads to changes in the levels of a number of synaptic proteins as well as changes in the levels of secreted amyloid beta (Aβ) 42 peptide and Tau phosphorylation. Finally, we report that local decrease in PREPL levels in mouse hippocampus attenuates long-term potentiation, suggesting a role in synaptic plasticity. Together, our results indicate that PREPL affects neuronal function by modulating protein trafficking and synaptic function, an important mechanism of AD pathogenesis. SIGNIFICANCE STATEMENT: Integrative network analysis reveals proline endopeptidase-like protein (PREPL) to be downregulated in human sporadic late-onset Alzheimer's disease brains. Down regulation of PREPL leads to increases in amyloid beta secretion, Tau phosphorylation, and decreases in protein trafficking and long-term potentiation.

Disulfiram with Cu2+ alleviates dextran sulfate sodium-induced ulcerative colitis in mice.

Background: Disulfiram (DSF), a Food and Drug Administration (FDA)-approved drug for chronic alcohol addiction, has anti-inflammatory effects that help prevent various cancers, and Cu2+ can enhance the effects of DSF. Inflammatory bowel diseases (IBD) are characterized by chronic or recurrent relapsing gastrointestinal inflammation. Many drugs targeting the immune responses of IBD have been developed, but their application has many problems, including side effects and high costs. Therefore, there is an urgent need for new drugs. In this study, we investigated the preventive effects of DSF+Cu2+ on dextran sulfate sodium (DSS)-induced ulcerative colitis (UC) in mice. Methods: The anti-inflammatory effects were investigated using the DSS-induced colitis mouse model and lipopolysaccharide (LPS)-induced macrophages. DSS-induced TCRβ-/- mice were used to demonstrate the effect of DSF in conjunction with Cu2+ on CD4+ T cell-secreted interleukin 17 (IL-17). In addition, the effect of DSF+Cu2+ on intestinal flora was studied by 16S rRNA microflora sequencing. Results: DSF and Cu2+ could significantly reverse the symptom of DSS-induced UC in mice, such as weight loss, disease activity index score, colon length shortening, and reversal of colon pathological changes. DSF and Cu2+ could inhibit colonic macrophage activation by blocking the nuclear factor kappa B (NF-κB) pathway, reducing nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing 3 (NLRP3)-inflammasome-derived interleukin 1 beta (IL-1β) secretion and caspase-1 (CASP1) activation, and decreasing IL-17 secretion by CD4+ T cells. Moreover, the treatment of DSF and Cu2+ could protect the intestinal barrier by reversing the expression of tight junction proteins, zonula occluden-1 (ZO-1), occludin, and mucoprotein-2 (MUC2). Additionally, DSF+Cu2+ could reduce the abundance of harmful bacteria and increase beneficial bacteria in the intestinal tract of mice, effectively improving intestinal microecology. Conclusion: Our study evaluated the effect of DSF+Cu2+ on the immune system and gut microbiota in colonic inflammation and highlighted its potential to treat UC in the clinic.

SPS, a sulfated galactoglucan of Laetiporus sulphureus, exhibited anti-inflammatory activities

Laetiporus sulphureus is an edible and medicinal mushroom. A sulfated galactoglucan (SPS) was isolated by the papain method. Polysaccharides (PS) were isolated by hot water and ethanol precipitation. The medium molecular weight SPS of 100 to 1000 kDa accounted for over half of the SPS mixture. Fucose, galactose, glucose, and mannose were the major monosaccharides in SPS and PS. The amount of sulfate in SPS was 1.09 mmol/g. SPS showed inhibition of tumor necrosis factor-α (TNF-α) release and reversed IκB degradation in LPS-induced RAW264.7 macrophages. The suppression of TNF-α secretion by SPS was through inhibiting the phosphorylation of AKT/extracellular signal-regulated kinases (ERK), p38, and c-Jun N-terminal kinase (JNK). A purified SPS, named SPS-3, was proven to inhibit the LPS-induced phosphorylation of AKT, ERK, and p-38 in RAW264.7 cells. The suppression of interleukin 6 (IL-6) and transforming growth factor beta (TGFβ) secretion by PS was through inhibiting LPS-induced phosphorylation of p-38 and TGF-β receptor II (TGFRII) signaling pathways. This study demonstrates that the isolated SPS and PS from L. sulphureus possessed good anti-inflammatory activity for dietary supplements and functional food.

Role of metabolic reprogramming in pro-inflammatory cytokine secretion from LPS or silica-activated macrophages.

In the lungs, macrophages constitute the first line of defense against pathogens and foreign bodies and play a fundamental role in maintaining tissue homeostasis. Activated macrophages show altered immunometabolism and metabolic changes governing immune effector mechanisms, such as cytokine secretion characterizing their classic (M1) or alternative (M2) activation. Lipopolysaccharide (LPS)-stimulated macrophages demonstrate enhanced glycolysis, blocked succinate dehydrogenase (SDH), and increased secretion of interleukin-1 beta (IL-1β) and tumor necrosis factor-alpha (TNF-α). Glycolysis suppression using 2 deoxyglucose in LPS-stimulated macrophages inhibits IL-1β secretion, but not TNF-α, indicating metabolic pathway specificity that determines cytokine production. In contrast to LPS, the nature of the immunometabolic responses induced by non-organic particles, such as silica, in macrophages, its contribution to cytokine specification, and disease pathogenesis are not well understood. Silica-stimulated macrophages activate pattern recognition receptors (PRRs) and NLRP3 inflammasome and release IL-1β, TNF-α, and interferons, which are the key mediators of silicosis pathogenesis. In contrast to bacteria, silica particles cannot be degraded, and the persistent macrophage activation results in an increased NADPH oxidase (Phox) activation and mitochondrial reactive oxygen species (ROS) production, ultimately leading to macrophage death and release of silica particles that perpetuate inflammation. In this manuscript, we reviewed the effects of silica on macrophage mitochondrial respiration and central carbon metabolism determining cytokine specification responsible for the sustained inflammatory responses in the lungs.

Membrane Bound CRT Fragment Accelerates Tumor Growth of Melanoma B16 Cell In Vivo through Promoting M2 Polarization via TLR4.

Calreticulin (CRT) is a major calcium-binding luminal resident protein on the endoplasmic reticulum that can also be released extracellular as well as anchored on surface of cells. Previously, we demonstrated that soluble recombinant CRT fragment 39-272 (CRT/39-272) exhibited potent immunostimulatory effects as well as immunoregulation effects on immune cells. Here, we constructed stable B16 melanoma cell lines expressing recombinant CRT/39-272 on the membrane (B16-tmCRT/39-272) to investigate the roles of cell surface CRT on tumor progression. We found that B16-tmCRT/39-272 cells subcutaneously inoculated into C57BL/6 mice exhibited stronger tumorigenicity than the B16-EGFP control cells. The tumor associated macrophages infiltrated in tumors were mainly M2 phenotype. Regulatory T cells (Tregs) were also expanded more in bearing mice. Consistent with the in vivo results, B16-tmCRT/39-272 promoted macrophage polarization toward F4/80+CD206+ M2 macrophages and promoted transforming growth factor beta (TGF-β) secretion in vitro, which could promote naïve CD4+T cell differentiation into Tregs. These results imply that the tmCRT/39-272 could accelerate tumor development by enhancing M2 macrophage polarization to induce TGF-β secretion, and then promoted Treg differentiation in the tumor microenvironment. Our data may provide useful clues for better understanding of the potentiating roles of CRT in tumorigenesis.

The interplay between serine proteases and caspase-1 regulates the autophagy-mediated secretion of Interleukin-1 beta in human neutrophils.

Neutrophils play major roles against bacteria and fungi infections not only due to their microbicide properties but also because they release mediators like Interleukin-1 beta (IL-1β) that contribute to orchestrate the inflammatory response. This cytokine is a leaderless protein synthesized in the cytoplasm as a precursor (pro-IL-1β) that is proteolytically processed to its active isoform and released from human neutrophils by secretory autophagy. In most myeloid cells, pro-IL-1β is processed by caspase-1 upon inflammasome activation. Here we employed neutrophils from both healthy donors and patients with a gain-of-function (GOF) NLRP3-mutation to dissect IL-1β processing in these cells. We found that although caspase-1 is required for IL-1β secretion, it undergoes rapid inactivation, and instead, neutrophil serine proteases play a key role in pro-IL-1β processing. Our findings bring to light distinctive features of the regulation of caspase-1 activity in human neutrophils and reveal new molecular mechanisms that control human neutrophil IL-1β secretion.

Immunoregulatory and neuroprotective activity of ovocystatin

IntroductionOvocystatin has beneficial properties for cognitive function in young rats and might prevents aging-related cognitive impairment in older animals, as well as reduces memory decline in APP/PS1 mice model.ObjectivesOur study aimed at assessing the impact of ovocystatin on microglia activation and neurogenesis.MethodsImmunoactivation: Mouse wild type microglia were stimulated with ovocystatin at dose of 100 micrograms/ml. The effect of ovocystatin on nitric oxide production and interleukin 1 beta secretion were determined. Neurogenesis: Primary rat hippocampal neurons of H19-7 cell line was used. The impact of ovocystatin on proliferation, nitric oxide production, and expression of markers of neurogenesis: microtubule-associated protein 2 (MAP2, isoforms A/B and C/D) and Synapsin 1, were determined.ResultsIt was shown that ovocystatin does not stimulate microglial cells to produce inflammatory mediators. Whereas, no toxic effect of ovocystatin (1-100 ug/ml) on H19-7 cells viability, and dose-dependent down-regulation of proliferation were demonstrated. It was also shown that in primary hippocampal neurons of H19-7 cells incubated with ovocystatin (100 micrograms/ml), the expression level of MAP2 C/D (75kDa) - characteristic form of immature neurons is unchanged. However, the increased expression of MAP2 A/B protein (280 kDa) – characteristic for mature neurons was observed after 6 and 24h incubation with ovocystatin. Relatively to MAP2 A/B, increased expression of synapsin 1 was observed.ConclusionsThe ovocystatin might be a potential activator of molecular mechanisms in primary hippocampal neurons, participating in regulation of neurogenesis. Nevertheless, further studies are needed.DisclosureNo significant relationships.

Comparable transforming growth factor beta-mediated immune suppression in ex vivo-expanded natural killer cells from cord blood and peripheral blood: implications for adoptive immunotherapy

T cell-based therapies like genetically modified immune cells expressing chimeric antigen receptors have shown robust anti-cancer activity in vivo, especially in patients with blood cancers. However, extending this approach to an “off-the-shelf” setting can be challenging, as allogeneic T cells carry a significant risk of graft-versus-host disease (GVHD). By contrast, allogeneic natural killer (NK) cells recognize malignant cells without the need for prior antigen exposure and have been used safely in multiple cancer settings without the risk of GVHD. However, similar to T cells, NK cell function is negatively impacted by tumor-induced transforming growth factor beta (TGF-β) secretion, which is a ubiquitous and potent immunosuppressive mechanism employed by most malignancies. Allogeneic NK cells for adoptive immunotherapy can be sourced from peripheral blood (PB) or cord blood (CB), and the authors’ group and others have previously shown that ex vivo expansion and gene engineering can overcome CB-derived NK cells’ functional immaturity and poor cytolytic activity, including in the presence of exogenous TGF-β. However, a direct comparison of the effects of TGF-β-mediated immune suppression on ex vivo-expanded CB- versus PB-derived NK cell therapy products has not previously been performed. Here the authors show that PB- and CB-derived NK cells have distinctive gene signatures that can be overcome by ex vivo expansion. Additionally, exposure to exogenous TGF-β results in an upregulation of inhibitory receptors on NK cells, a novel immunosuppressive mechanism not previously described. Finally, the authors provide functional and genetic evidence that both PB- and CB-derived NK cells are equivalently susceptible to TGF-β-mediated immune suppression. The authors believe these results provide important mechanistic insights to consider when using ex vivo-expanded, TGF-β-resistant PB- or CB-derived NK cells as novel immunotherapy agents for cancer.

Modified lipid metabolism and cytosolic phospholipase A2 activation in mesangial cells under pro-inflammatory conditions.

Diabetic kidney disease is a consequence of hyperglycemia and other complex events driven by early glomerular hemodynamic changes and a progressive expansion of the mesangium. The molecular mechanisms behind the pathophysiological alterations of the mesangium are yet to be elucidated. This study aimed at investigating whether lipid signaling might be the missing link. Stimulation of human mesangial cells with high glucose primed the inflammasome-driven interleukin 1 beta (IL-1β) secretion, which in turn stimulated platelet-derived growth factor (PDGF-BB) release. Finally, PDGF-BB increased IL-1β secretion synergistically. Both IL-1β and PDGF-BB stimulation triggered the formation of phosphorylated sphingoid bases, as shown by lipidomics, and activated cytosolic phospholipase cPLA2, sphingosine kinase 1, cyclooxygenase 2, and autotaxin. This led to the release of arachidonic acid and lysophosphatidylcholine, activating the secretion of vasodilatory prostaglandins and proliferative lysophosphatidic acids. Blocking cPLA2 release of arachidonic acid reduced mesangial cells proliferation and prostaglandin secretion. Validation was performed in silico using the Nephroseq database and a glomerular transcriptomic database. In conclusion, hyperglycemia primes glomerular inflammatory and proliferative stimuli triggering lipid metabolism modifications in human mesangial cells. The upregulation of cPLA2 was critical in this setting. Its inhibition reduced mesangial secretion of prostaglandins and proliferation, making it a potential therapeutical target.