IL-1β In Response Research Articles

Role of scavenger receptors in silica nanoparticle-induced cytokine responses in bronchial epithelial cells

A major challenge in nanoparticle (NP) research is to elucidate how NPs activate initial targets in cells, leading to cytotoxicity and inflammation. We have previously shown that silica (Si)NPs induce pro-inflammatory responses in bronchial epithelial cells (BEAS-2B) via mechanisms involving transforming growth factor (TGF)-α release, and activation of MAP-kinase p38 and JNK besides NF-κB (p65). In the present study, the roles of scavenger receptors (SRs) in SiNP-induced cytokine responses in BEAS-2B cells were examined by siRNA silencing. Cells exposed to Si10 and Si50 (nominal sizes 10 and 50 nm) showed marked interleukin (IL)-6, CXCL8, IL-1α, IL-1β responses. Transient knockdown of SR-B1, LOX-1 and CXCL16 reduced the Si10- and Si50-induced cytokine responses, to a different magnitude dependent on the particle size, SR and cytokine. Si10-induced TGF-α responses were also markedly reduced by knockdown of SR-B1 and CXCL16. Furthermore, the role of SR-B1 in Si10-induced phosphorylations of p65 and MAP-kinases p38 and JNK were examined, and no significant reductions were observed upon knockdown of SR-B1. In conclusion, LOX-1 and CXCL16 and especially SR-B1 seem to have important roles in mediating cytokine responses and TGF-α release due to SiNP exposure in BEAS-2B cells, without a down-stream role of MAP-kinase and NF-κB.

OTULIN maintains skin homeostasis by controlling keratinocyte death and stem cell identity

OTULIN is a deubiquitinase that specifically cleaves linear ubiquitin chains. Here we demonstrate that the ablation of Otulin selectively in keratinocytes causes inflammatory skin lesions that develop into verrucous carcinomas. Genetic deletion of Tnfr1, knockin expression of kinase-inactive Ripk1 or keratinocyte-specific deletion of Fadd and Mlkl completely rescues mice with OTULIN deficiency from dermatitis and tumorigenesis, thereby identifying keratinocyte cell death as the driving force for inflammation. Single-cell RNA-sequencing comparing non-lesional and lesional skin reveals changes in epidermal stem cell identity in OTULIN-deficient keratinocytes prior to substantial immune cell infiltration. Keratinocytes lacking OTULIN display a type-1 interferon and IL-1β response signature, and genetic or pharmacologic inhibition of these cytokines partially inhibits skin inflammation. Finally, expression of a hypomorphic mutant Otulin allele, previously shown to cause OTULIN-related autoinflammatory syndrome in humans, induces a similar inflammatory phenotype, thus supporting the importance of OTULIN for restraining skin inflammation and maintaining immune homeostasis.

Aloe-Emodin-Mediated Photodynamic Therapy Attenuates Sepsis-Associated Toxins in Selected Gram-Positive Bacteria In Vitro.

Sepsis is an acute inflammatory response that leads to life-threatening complications if not quickly and adequately treated. Cytolysin, hemolysin, and pneumolysin are toxins produced by gram-positive bacteria and are responsible for resistance to antimicrobial drugs, cause virulence and lead to sepsis. This work assessed the effects of aloe-emodin (AE) and photodynamic therapy (PDT) on sepsis-associated gram-positive bacterial toxins. Standard and antibiotic-resistant Enterococcus faecalis, Staphylococcus aureus, and Streptococcus pneumonia bacterial strains were cultured in the dark with varying AE concentrations and later irradiated with 72 J/cm-2 light. Colony and biofilm formation was determined. CCK-8, Griess reagent reaction, and ELISA assays were done on bacteria-infected RAW264.7 cells to determine the cell viability, NO, and IL-1β and IL-6 pro-inflammatory cytokines responses, respectively. Hemolysis and western blot assays were done to determine the effect of treatment on hemolysis activity and sepsis-associated toxins expressions. AE-mediated PDT reduced bacterial survival in a dose-dependent manner with 32 μg/ml of AE almost eliminating their survival. Cell proliferation, NO, IL-1β, and IL-6 cytokines production were also significantly downregulated. Further, the hemolytic activities and expressions of cytolysin, hemolysin, and pneumolysin were significantly reduced following AE-mediated PDT. In conclusion, combined use of AE and light (435 ± 10 nm) inactivates MRSA, S. aureus (ATCC 29213), S. pneumoniae (ATCC 49619), MDR-S. pneumoniae, E. faecalis (ATCC 29212), and VRE (ATCC 51299) in an AE-dose dependent manner. AE and light are also effective in reducing biofilm formations, suppressing pro-inflammatory cytokines, hemolytic activities, and inhibiting the expressions of toxins that cause sepsis.

Porphyromonas Gingivalis Infection Induces Synaptic Failure via Increased IL-1β Production in Leptomeningeal Cells.

Studies have reported that synaptic failure occurs before the Alzheimer's disease (AD) onset. The systemic Porphyromonas gingivalis (P. gingivalis) infection is involved in memory decline. We previously showed that leptomeningeal cells, covering the brain, activate glial cells by releasing IL-1β in response to systemic inflammation. In the present study, we focused on the impact of leptomeningeal cells on neurons during systemic P. gingivalis infection. The responses of leptomeningeal cells and cortical neurons to systemic P. gingivalis infection were examined in 15-month-old mice. The mechanism of IL-1β production by P. gingivalis infected leptomeningeal cells was examined, and primary cortical neurons were treated with P. gingivalis infected leptomeningeal cells condition medium (Pg LCM). Systemic P. gingivalis infection increased the expression of IL-1β in leptomeninges and reduced the synaptophysin (SYP) expression in leptomeninges proximity cortex in mice. Leptomeningeal cells phagocytosed P. gingivalis resulting in lysosomal rupture and cathepsin B (CatB) leakage. Leaked CatB mediated NLRP3 inflammasome activation inducing IL-1β secretion in leptomeningeal cells. Pg LCM decreased the expression of synaptic molecules, including SYP, which was inhibited by an IL-1 receptor antagonist pre-treatment. These observations demonstrate that P. gingivalis infection is involved in synaptic failure by inducing CatB/NLRP3 inflammasome-mediated IL-1β production in leptomeningeal cells. The periodontal bacteria-induced synaptic damage may accelerate the onset and cognitive decline of AD.

Rapid Release of Interleukin-1β from Human Platelets Is Independent of NLRP3 and Caspase.

Platelets are critical in mediating both rapid responses to injury and the development and progression of coronary disease. Several studies have shown that, after prolonged exposure to agonists, they produce and release inflammatory mediators including interleukin-1β (IL-1β), via the classical pathway (NLRP3 inflammasome and caspase-1 cleavage to release active IL-1β) as described for leukocytes. This study aimed to determine whether there is rapid release of IL-1β in response to soluble platelet agonists and whether such rapid release is NLRP3- and caspase-1-dependent. Using flow cytometry to detect platelet activation (and release of α and dense granule contents) and the combination of Western blotting, enzyme-linked-immunosorbent assay, and immunogold labeling transmission electron and immunofluorescence microscopy, we identified that resting human platelets contain mature IL-1β. Platelets release IL-1β within minutes in response to adenosine diphosphate (ADP), collagen, and thrombin receptor agonists, but not in response to conventional NLRP3 inflammasome agonists-lipopolysaccharide and adenosine triphosphate. The rapid release of IL-1β in response to ADP and thrombin receptor agonists was independent of caspases (including caspase-1) and NLRP3. Immature and mature IL-1β were identified as low-abundance proteins on transmission electron microscopy of human platelets, and were localized to the platelet cytosol, open canalicular system, and the periphery of α granules. Unlike monocytes and neutrophils, human platelets are capable of rapid agonist- and time-dependent release of IL-1β by a mechanism which is independent of caspase-1 and NLRP3.

Children With Asthma Have Impaired Innate Immunity and Increased Numbers of Type 2 Innate Lymphoid Cells Compared With Healthy Controls

BackgroundAsthma is the most frequent cause of hospitalisation among children; however, little is known regarding the effects of asthma on immune responses in children.ObjectiveThe present study aimed to evaluate cytokine responses of peripheral blood mononuclear cells (PBMCs), PBMC composition and lung function in children with and without asthma.MethodsUsing a case-control design, we compared 48 children with asthma aged 3-11 years with 14 age-matched healthy controls. PBMC composition and cytokine production including interferon (IFN)-γ, interleukin (IL)-1β, IL-5 and lL-6 following stimulation with rhinovirus-1B (RV1B), house dust mite (HDM) and lipopolysaccharide (LPS) were measured. Lung function was assessed using impulse oscillometry and nitrogen multiple breath washout.ResultsThe frequency of group 2 innate lymphoid cells were significantly higher in asthmatics and PBMCs from asthmatics had deficient IFN-γ production in response to both RV1B and LPS compared with controls (P<0.01). RV1B-induced IL-1β response and HDM-stimulated IL-5 production was higher in asthmatics than controls (P<0.05). In contrast, IL-1β and IL-6 were significantly reduced in response to HDM and LPS in asthmatics compared to controls (P<0.05). Children with asthma also had reduced pulmonary function, indicated by lower respiratory reactance as well as higher area of-reactance and lung clearance index values compared with controls (P<0.05).ConclusionOur study indicates that children with asthma have a reduced lung function in concert with impaired immune responses and altered immune cell subsets. Improving our understanding of immune responses to viral and bacterial infection in childhood asthma can help to tailor management of the disease.

The Divergent Immunomodulatory Effects of Short Chain Fatty Acids and Medium Chain Fatty Acids.

Fatty acids are derived from diet and fermentative processes by the intestinal flora. Two to five carbon chain fatty acids, termed short chain fatty acids (SCFA) are increasingly recognized to play a role in intestinal homeostasis. However, the characteristics of slightly longer 6 to 10 carbon, medium chain fatty acids (MCFA), derived primarily from diet, are less understood. Here, we demonstrated that SCFA and MCFA have divergent immunomodulatory propensities. SCFA down-attenuated host pro-inflammatory IL-1β, IL-6, and TNFα response predominantly through the TLR4 pathway, whereas MCFA augmented inflammation through TLR2. Butyric (C4) and decanoic (C10) acid displayed most potent modulatory effects within the SCFA and MCFA, respectively. Reduction in TRAF3, IRF3 and TRAF6 expression were observed with butyric acid. Decanoic acid induced up-regulation of GPR84 and PPARγ and altered HIF-1α/HIF-2α ratio. These variant immune characteristics of the fatty acids which differ by just several carbon atoms may be attributable to their origins, with SCFA being primarily endogenous and playing a physiological role, and MCFA exogenously from the diet.

Acute systemic inflammation exacerbates neuroinflammation in Alzheimer's disease: IL-1β drives amplified responses in primed astrocytes and neuronal network dysfunction.

Neuroinflammation contributes to Alzheimer's disease (AD) progression. Secondary inflammatory insults trigger delirium and can accelerate cognitive decline. Individual cellular contributors to this vulnerability require elucidation. Using APP/PS1 mice and AD brain, we studied secondary inflammatory insults to investigate hypersensitive responses in microglia, astrocytes, neurons, and human brain tissue. The NLRP3 inflammasome was assembled surrounding amyloid beta, and microglia were primed, facilitating exaggerated interleukin-1β(IL-1β) responses to subsequent LPS stimulation. Astrocytes were primed to produce exaggerated chemokine responses to intrahippocampal IL-1β. Systemic LPS triggered microglial IL-1β, astrocytic chemokines, IL-6, and acute cognitive dysfunction, whereas IL-1β disrupted hippocampal gamma rhythm, all selectively in APP/PS1 mice. Brains from AD patients with infection showed elevated IL-1β and IL-6 levels. Therefore, amyloid leaves the brain vulnerable to secondary inflammation at microglial, astrocytic, neuronal, and cognitive levels, and infection amplifies neuroinflammatory cytokine synthesis in humans. Exacerbation of neuroinflammation to produce deleterious outcomes like delirium and accelerated disease progression merits careful investigation in humans.

Brucella Outer Membrane Lipoproteins 19 and 16 Differentially Induce Interleukin-18 Response or Pyroptosis in Human Monocytic Cells.

Brucella species are Gram-negative intracellular bacteria that causes severe inflammatory diseases in animals and humans. Two major lipoproteins (L19 and L16) of Brucella outer membrane proteins were studied to explore the association with inflammatory response of human monocytes (THP-1). Activated THP-1 cells induced with recombinant L19 and L16 were analyzed in comparison with unlipidated forms (U19 and U16) and lipopolysaccharide (LPS) of Brucella melitensis, respectively. Secretion of inflammatory factors tumor necrosis factor-α, interleukin (IL)-6, and IL-1β was significantly increased from L19, L16, or both stimulated THP-1 cells. High secretion of IL-18 was detected only from L19-induced cells. Signaling of those cytokine responses was identified mainly through the P38-mitogen-activated protein kinase pathway, and signaling of L19-induced IL-1β response partly occurred via necrosis factor-κB. While exploring different forms of IL-18, we found that L19-induced production of active IL-18 (18 kD) occurred through upregulating NLRP3 and activating caspase-1, whereas L16-induced production of inactive IL-18 fragments (15 kD and 16 kD) occurred through activating caspase-8/3. We also found that L19 upregulated phosphorylation of XIAP for inhibiting caspase-3 activity to cleave IL-18, whereas L16 activated caspase-3 for producing GSDME-N and leading to pyroptosis of THP-1 cells. Brucella L19 and L16 differentially induce IL-18 response or pyroptosis in THP-1 cells, respectively.

Interleukin-1β exacerbates disease and is a potential therapeutic target to reduce pulmonary inflammation during severe influenza A virus infection.

Hyperinflammatory responses including the production of NLRP3‐dependent interleukin (IL)‐1β is a characteristic feature of severe and fatal influenza A virus (IAV) infections. The NLRP3 inflammasome has been shown to play a temporal role during severe IAV immune responses, with early protective and later detrimental responses. However, the specific contribution of IL‐1β in modulating IAV disease in vivo is currently not well defined. Here, we identified that activation of NLRP3‐dependent IL‐1β responses occurs rapidly following HKx31 H3N2 infection, prior to the onset of severe IAV disease. Mature IL‐1β was detectable in vivo in both hemopoietic and nonhemopoietic cells. Significantly, therapeutic inhibition of IL‐1β in the airways with intranasal anti‐IL‐1β antibody treatment from day 3 postinfection, corresponding to the onset of clinical signs of disease, significantly prolonged survival and reduced inflammation in the airways. Importantly, early targeting of IL‐1β from day 1 postinfection also improved survival. Together, these studies specifically define a role for IL‐1β in contributing to the development of hyperinflammation and disease and indicate that targeting IL‐1β is a potential therapeutic strategy for severe IAV infections.

DmLT Adjuvant Enhances Cytokine Responses to T Cell Stimuli, Whole Cell Vaccine Antigens and Lipopolysaccharide in Both Adults and Infants.

Enhancement of mucosal immune responses in children and infants using novel adjuvants such as double mutant heat labile toxin (dmLT) is an important goal in the enteric vaccine field. dmLT has been shown to enhance mucosal IgA responses to the oral inactivated enterotoxigenic Escherichia coli (ETEC) vaccine ETVAX. dmLT can enhance IL-17A production from adult T cells, which may increase the production and secretion of mucosal IgA antibodies. However, the adjuvant mechanism remains to be fully elucidated and might differ between infants and adults due to age-related differences in the development of the immune system. The main objective of this study was to determine how dmLT influences antigen presenting cells and T cells from infants compared to adults, and the role of IL-1β for mediating the adjuvant activity. Peripheral blood mononuclear cells (PBMCs) from Bangladeshi infants (6-11 months) and adults (18-40 years) were stimulated with the mitogen phytohaemagglutinin (PHA), the superantigen Staphylococcal enterotoxin B (SEB), ETVAX whole cell component (WCC) or E. coli lipopolysaccharide (LPS) ± dmLT, and cytokine production was measured using ELISA and electrochemiluminescence assays. The adjuvant dmLT significantly enhanced SEB- and PHA-induced IL-17A, but not IFN-γ responses, in PBMCs from both infants and adults. Blocking experiments using an IL-1 receptor antagonist demonstrated the importance of IL-1 signaling for the adjuvant effect. dmLT, ETVAX WCC and LPS induced dose-dependent IL-1β responses of comparable magnitudes in infant and adult cells. Depletion experiments suggested that IL-1β was mainly produced by monocytes. dmLT enhanced IL-1β responses to low doses of WCC and LPS, and the adjuvant effect appeared over a wider dose-range of WCC in infants. dmLT and WCC also induced IL-6, IL-23 and IL-12p70 production in both age groups and dmLT tended to particularly enhance IL-23 responses to WCC. Our results show that dmLT can induce IL-1β as well as other cytokines, which in turn may enhance IL-17A and potentially modulate other immunological responses in both infants and adults. Thus, dmLT may have an important function in promoting immune responses to the ETVAX vaccine, as well as other whole cell- or LPS-based vaccines in infants in low- and middle-income countries.

Lung immune tone via gut-lung axis: gut-derived LPS and short-chain fatty acids' immunometabolic regulation of lung IL-1β, FFAR2, and FFAR3 expression.

Microbial metabolites produced by the gut microbiome, e.g. short-chain fatty acids (SCFA), have been found to influence lung physiology and injury responses. However, how lung immune activity is regulated by SCFA is unknown. We examined fresh human lung tissue and observed the presence of SCFA with interindividual variability. In vitro, SCFA were capable of modifying the metabolic programming in LPS-exposed alveolar macrophages (AM). We hypothesized that lung immune tone could be defined by baseline detection of lung intracellular IL-1β. Therefore, we interrogated naïve mouse lungs with intact gut microbiota for IL-1β mRNA expression and localized its presence within alveolar spaces, specifically within AM subsets. We established that metabolically active gut microbiota, which produce SCFA, can transmit LPS and SCFA to the lung and thereby could create primed lung immunometabolic tone. To understand how murine lung cells sensed and upregulated IL-1β in response to gut microbiome-derived factors, we determined that, in vitro, AM and alveolar type II (AT2) cells expressed SCFA receptors, free fatty acid receptor 2 (FFAR2), free fatty acid receptor 3 (FFAR3), and IL-1β but with distinct expression patterns and different responses to LPS. Finally, we observed that IL-1β, FFAR2, and FFAR3 were expressed in isolated human AM and AT2 cells ex vivo, but in fresh human lung sections in situ, only AM expressed IL-1β at rest and after LPS challenge. Together, this translational study using mouse and human lung tissue and cells point to an important role for the gut microbiome and their SCFA in establishing and regulating lung immune tone.

Production of IL-1β, IL-6 and CXCL8 by endometrium of crossbred heifers stimulated with various pathogen-associated molecular patterns

Uterine bacterial infections are common during the post-partum period of dairy herds and, apparently, incidences in crossbred cattle are less than in Holsteins. The aims of this study were (I) to evaluate production of interleukin 1-β (IL-1β), interleukin-6 (IL-6) and chemokine CXCL8 using endometrial explants from Bos indicus crossbred heifers at diestrous, stimulated by various pathogen-associated molecular patterns (PAMP), and (II) assess production of these cytokines by lipopolysaccharide-stimulated endometrial explants from heifers when samples were collected at different stages of estrous cycle. In the first experiment, endometrial explants from heifers at diestrous were stimulated by ten-fold serial dilutions of lipopolysaccharide (LPS), triacylated lipopeptide (PAM3) or peptidoglycan (PGN). In the second experiment, endometrial explants collected at different stages of the estrous cycle were treated with LPS. Concentrations of IL-1β, IL-6 and CXCL8 were quantified in supernatant. There was a marked (P < 0.05) production of IL-1β, IL-6, and CXCL8 in response to LPS treatment. There was also production of IL-1β (P < 0.05) in response to PGN treatment. Explant samples collected at different stages of the estrous cycle responded to LPS treatment with production of IL-1β and IL-6, but with no differences (P > 0.05) between stages of estrous cycle. In conclusion, endometrial samples of crossbred Zebu-based heifers collected during diestrous produced IL-1β, IL-6 and CXCL8 in response to LPS and IL-1β in response to PGN. The cytokine production in response to LPS, however, was not affected by the stage of the estrous cycle in Bos indicus crossbred heifers.

273 Bacterial components induce fetal membrane non-pyroptotic IL-1β production through activation of the NLRP3 inflammasome

Bacterial infections increase the risk of chorioamnionitis and preterm birth; however the mechanisms involved are not fully understood. Inflammatory interleukin-1β (IL-1β) is an important mediator of preterm birth, and its secretion is mediated by the inflammasome that processes pro-IL-1β into its active form. This study sought to determine the fetal membrane (FM) compartment involved in IL-1β production in response to bacterial components and to evaluate the mechanism of inflammasome activation. An ex vivo human FM explant system was used to evaluate the response of the amnion, chorion, and intact FMs to media or the bacterial components lipopolysaccharide (LPS), peptidoglycan (PGN), flagellin, iE-DAP, or MDP (n=8). After 24 hours, IL-1β secretion was quantified by ELISA, the pyroptotic marker Gasdermin-D (GSDMD) evaluated by Western blot, and viability measured using a LDH assay. Inflammasome function was assessed using inhibitors to NLRP3 (MCC950), caspase-1 (Z-WEHD-FMK), Pannexin-1 (carbenoxolone), and ROS (DPI) (n=8-12). LPS, PGN, and MDP significantly increased IL-1β secretion from intact FM explants by 57.0±22.4-; 38.3±16.4-; and 28.4±11.9-fold respectively (p<0.05). Flagellin and iE-DAP had no significant effect. The chorion, not the amnion, was the source of this IL-1β response. Inhibitors to NLRP3, Pannexin-1, and ROS significantly inhibited LPS, PGN, and MDP-induced IL-1β secretion from intact FM explants (p<0.05), while the caspase-1 inhibitor significantly reduced LPS and MDP, but not PGN-induced, IL-1β (p<0.05). There was no significant difference in GSDMD activity or LDH secretion by intact FMs under all treatment conditions. The chorion is the major source of IL-1β production following FM exposure to bacterial components. Bacterial LPS, PGN, and MDP induced human FM IL-1β secretion through activation of the NLRP3 inflammasome with contributions from ATP release through Pannexin-1 and ROS signaling. Further investigation is needed to determine the role of alternate caspases and non-pyroptotic mechanisms of FM IL-1β production and release.

Impact of mono-culture vs. Co-culture of keratinocytes and monocytes on cytokine responses induced by important skin sensitizers

Sensitization to a contact allergen brings with it a lifelong risk to develop allergic contact dermatitis. Inflammation is an important part of the skin sensitizing mechanism, and understanding how different haptens stimulate the immune system, as well as the role played by different cell types present in skin, may be helpful for developing optimized in vitro models for risk assessment of new chemicals or mixtures. The aim of this study was to compare the cytokine profile following exposure of cells representing keratinocytes (HaCaT), monocytes (THP-1) and a co-culture of these cells to three clinically important skin sensitizers: cobalt (II) chloride (CoCl2), methylisothiazolinone (MI) and p-phenylenediamine (PPD). Secretion of ten pro-inflammatory cytokines was measured using multiplexing. The results showed that the cytokine response differed substantially between the three cell assays. CoCl2 caused an increase of IL-8 in HaCaT cells, while the induction of also IL-13 and IL-1β was observed in THP-1 cells and co-cultures. MI induced six cytokines in HaCaT cells but only IL-1β in the THP-1 cells and four cytokines in the co-culture. Interestingly, the IL-1β response was massive in the co-culture. PPD caused release of IL-1β in all three models as well as IL-8 in the co-culture. Control experiments with two non-sensitizers and irritants (lactic acid and sodium dodecyl sulfate) showed no effect on IL-8 or IL-1β in the co-culture. Taken together, results from this exploratory analysis show unique cytokine profiles dependent on the type of hapten and cell model. Importantly, all three haptens triggered secretion of IL-1β and IL-8 in a co-culture of HaCaT cells and THP-1 cells, representing the most robust test system.

Downregulation of MicroRNA-495 Alleviates IL-1β Responses among Chondrocytes by Preventing SOX9 Reduction.

PurposeOur previous work demonstrated that miRNA-495 targets SOX9 to inhibit chondrogenesis of mesenchymal stem cells. In this study, we aimed to investigate whether miRNA-495-mediated SOX9 regulation could be a novel therapeutic target for osteoarthritis (OA) using an in vitro cell culture model.Materials and MethodsAn in vitro model mimicking the OA environment was established using TC28a2 normal human chondrocyte cells. Interleukin-1β (IL-1β, 10 ng/mL) was utilized to induce inflammation-related changes in TC28a2 cells. Safranin O staining and glycosaminoglycan assay were used to detect changes in proteoglycans among TC28a2 cells. Expression levels of COX-2, ADAMTS5, MMP13, SOX9, CCL4, and COL2A1 were examined by qRT-PCR and/or Western blotting. Immunohistochemistry was performed to detect SOX9 and CCL4 proteins in human cartilage tissues obtained from patients with OA.ResultsmiRNA-495 was upregulated in IL-1β-treated TC28a2 cells and chondrocytes from damaged cartilage tissues of patients with OA. Anti-miR-495 abolished the effect of IL-1β in TC28a2 cells and rescued the protein levels of SOX9 and COL2A1, which were reduced by IL-1β. SOX9 was downregulated in the damaged cartilage tissues of patients with OA, and knockdown of SOX9 abolished the effect of anti-miR-495 on IL-1β-treated TC28a2 cells.ConclusionWe demonstrated that inhibition of miRNA-495 alleviates IL-1β-induced inflammatory responses in chondrocytes by rescuing SOX9 expression. Accordingly, miRNA-495 could be a potential novel target for OA therapy, and the application of anti-miR-495 to chondrocytes could be a therapeutic strategy for treating OA.

Identification of cell-surface glycans that mediate motility-dependent binding and internalization of Pseudomonas aeruginosa by phagocytes

Phagocytic cells are critical to host defense against Pseudomonas aeruginosa, a Gram-negative bacterium that is an opportunistic pathogen. Accordingly, susceptible individuals frequently have impaired innate immune responses, including those with cystic fibrosis or neutropenia. Previous studies identified that the downregulation, or loss, of bacterial flagellar motility enables bacteria to evade interactions with phagocytic cells that result in phagocytic uptake of the bacteria. However, the mechanistic bases for motility-dependent interactions between P. aeruginosa and host cell surfaces that lead to phagocytic uptake of the bacteria are poorly understood. A recent insight is that exogenous addition of a negatively charged phospholipid, phosphatidylinositol-(3,4,5)-triphosphate (PIP3), promotes the engagement of non-motile strains of P. aeruginosa with phagocytes leading to uptake of the bacteria. Thus, we hypothesized that the engagement of P. aeruginosa by phagocytic cells is mediated by motility-dependent interactions with cell-surface polyanions. Here we report that endogenous polyanionic N-linked glycans and heparan sulfate mediate bacterial binding of P. aeruginosa by human monocytic cells. These specific interactions resulted in P. aeruginosa phagocytosis, bacterial type 3 secretion system (T3SS)-mediated cellular intoxication and the IL-1β response of host innate immune cells. Importantly, the bacterial interactions with the glycans were motility-dependent and could be recapitulated with purified, immobilized glycans. Therefore, this work describes novel interactions of P. aeruginosa with specific phagocyte cell-surface glycans that modulate relevant host innate immune responses to the bacteria, including phagocytosis, inflammation and cytotoxicity.

Transcriptional response modules characterize IL-1β and IL-6 activity in COVID-19.

SummaryDysregulated IL-1β and IL-6 responses have been implicated in the pathogenesis of severe Coronavirus Disease 2019 (COVID-19). Innovative approaches for evaluating the biological activity of these cytokines in vivo are urgently needed to complement clinical trials of therapeutic targeting of IL-1β and IL-6 in COVID-19. We show that the expression of IL-1β or IL-6 inducible transcriptional signatures (modules) reflects the bioactivity of these cytokines in immunopathology modelled by juvenile idiopathic arthritis (JIA) and rheumatoid arthritis. In COVID-19, elevated expression of IL-1β and IL-6 response modules, but not the cytokine transcripts themselves, is a feature of infection in the nasopharynx and blood but is not associated with severity of COVID-19 disease, length of stay, or mortality. We propose that IL-1β and IL-6 transcriptional response modules provide a dynamic readout of functional cytokine activity in vivo, aiding quantification of the biological effects of immunomodulatory therapies in COVID-19.

Prospective association of maternal immune pro-inflammatory responsivity and regulation in pregnancy with length of gestation.

The immune system represents a leading pathway of interest in the pathophysiology of preterm birth. The majority of human clinical studies interrogating this pathway have utilized circulating immune biomarkers; however, these concentrations typically reflect only basal production but not key functional properties of the immune system, particularly variation in the pro-inflammatory response to antigen challenge and the regulation of this response. Thus, in this study, we utilized an ex vivo stimulation protocol that quantifies these processes, and we examined their prospective association with the gestation length and risk of preterm birth. Immune responsiveness and regulation were assessed in 128 pregnant women in mid-gestation using an ex vivo stimulation protocol. Maternal pro-inflammatory responsivity of leukocytes was quantified by assessing the release of the pro-inflammatory cytokines IL-6, TNF-α, and IL-1β in response to antigen stimulation, and regulation of the pro-inflammatory response was quantified by assessing the suppression of stimulated cytokine response upon co-incubation with increasing dexamethasone concentrations (ie, glucocorticoid receptor resistance; GRR). Higher maternal GRR, indicating impaired regulation of the pro-inflammatory response, was significantly and independently associated with shorter gestational length (β=-0.42, p=.0091) and a 3.0-fold increase in risk for preterm birth (OR=3.01, 95% CI=1.17-7.70, p=.0218). Basal circulating IL-6 and TNF-α were not associated with either outcome. The association of maternal GRR with length of gestation and preterm birth risk suggests that the processes represented by this measure-maternal pro-inflammatory propensity and immune regulation-may provide further mechanistic insight into the pathophysiology of preterm birth.

Increased Monocyte Inflammatory Responses to Oxidized LDL Are Associated with Insulin Resistance in HIV-Infected Individuals on Suppressive Antiretroviral Therapy.

Despite long term antiretroviral therapy (ART), insulin resistance (IR) is common among people living with HIV/AIDS (PLWHA) exposing this population to a greater risk of cardiometabolic complications when compared to their uninfected counterparts. We previously identified an expansion in monocyte subpopulations in blood that were linked to the degree of IR in persons with HIV on stable ART. In this study, we directly assessed monocyte inflammatory functional properties from PLWHA on ART (n = 33) and HIV-uninfected controls (n = 14) of similar age, gender, and cardiovascular disease risk and determined the relationship with IR (homeostatic model assessment-insulin resistance (HOMA-IR)), calculated from fasting blood glucose and insulin measurements. Peripheral blood mononuclear cells were stimulated with oxidized low-density lipoproteins (oxLDL) and polyfunctional monocyte cytokine responses (IL-1β, IL-6, IL-8, or TNF-α) were determined by flow cytometry. Higher monocyte IL-1β and IL-8 responses to oxLDL were associated with higher IR in PLWHA but not in the control group. We observed that higher basal monocyte cytokine responses were associated with both duration since HIV diagnosis and ART initiation. In the management of IR in chronic HIV, strategies lowering monocyte IL-1β and IL-8 responses should be considered in addition to ART in order to limit adverse cardio-metabolic outcomes.