Swertiamarin ameliorates inflammation and osteoclastogenesis intermediates in IL-1β induced rat fibroblast-like synoviocytes

Objective and design

Rheumatoid arthritis is a chronic inflammatory and autoimmune disease that leads to aggressive joint cartilage and bone destruction. Swertiamarin is a secoiridoid glycoside found in Enicostema axillare (Lam) A. Raynal, a medicinal plant used in the Indian system of traditional medicine. In the present study, the potential of swertiamarin was evaluated in IL-1β induced fibroblast-like synoviocytes (FLS).

Methods

The FLS were isolated from Freund’s Complete Adjuvant induced arthritic (AA) rats and cultured with IL-1β. The normal FLS and AA-FLS were cultured and used for subsequent experiment in fibroblastic morphology form. The efficacy of swertiamarin (10–50 μg/ml) was evaluated on mRNA and protein expression levels of inflammatory and osteoclastogenesis mediators. The efficacy was also evaluated on p38 MAPKα levels with time course studies (2, 4, 6, 8 and 12 h).

Results

IL-1β induced cell proliferation (149.46 ± 13.73 %) and NO production (162.03 ± 11.03 %) in AA-FLS; treatment with swertiamarin controlled proliferation (82.77 ± 4.22 %) and NO production (82.06 ± 3.91 % at 50 μg/ml) in a dose-dependent manner. It also significantly (P < 0.05) modulated the expression of apoptotic marker (caspase 3), proinflammation mediators (TNFα, IL-6, PGE2, COX-2, iNOS, MMPs) and osteoclastogenic mediator (RANKL) at both the mRNA and protein levels. Treatment with swertiamarin inhibited the levels of p38 MAPKα in a dose-dependent manner and also significantly (P < 0.05) attenuated the release of the same in time dependent mode.

Conclusion

These findings suggest that treatment with swertiamarin attenuated IL-1β induced FLS, and it revealed anti-inflammatory potential by attenuating aggressive FLS.     

IL-1β induces expression of costimulatory molecules and cytokines but not immune feedback regulators in dendritic cells

Dendritic cells play an important role in the initiation of immune reactions. Due to their high capacity to prime T-cell responses, the activation of dendritic cells must be tightly controlled. Because Interleukin-1β (IL-1β) is a key player in autoinflammatory diseases, we compared the ability of IL-1β to activate human dendritic cells and induce immune-regulatory molecules versus the effects induced by pathogen-derived stimuli.Upon activation with either IL-1β or microbial stimuli, monocyte-derived dendritic cells showed enhanced expression of costimulatory molecules, increased secretion of chemokines and cytokines, and the ability to activate T cells. In contrast, immune-feedback molecules, including PD-L1, IL-1RA, IL-10 and SOCS1, were exclusively upregulated in response to microbial stimuli, whereas IL-1β treatment had no inducing effect on them. Thus, the limited capacity of IL-1β to induce potential feedback inhibitors may support its key etiologic role in chronic inflammation and autoinflammatory responses.     

Interleukin-1β induces expression of cyclooxygenase-2 mRNA in human gingival fibroblasts

The effect of interleukin-1 (IL-1) on the expression of cyclooxygenase-1 and –2 (COX-1 and COX-2) mRNA and its relation to prostaglandin E₂ (PGE₂) biosynthesis in human gingival fibroblasts was studied. IL-1 increased levels of mRNA for COX-2 whereas the COX-1 mRNA level was unaffected. The increased COX-2 mRNA levels were accompanied by enhanced PGE₂ formation. The phorbol, 12-myristate 13-acetate (PMA), known to stimulate protein kinase C (PKC), also induced expression of COX-2 mRNA. When gingival fibroblasts were treated simultaneously with IL-1 and PMA, the cytokine IL-1 synergistically increased levels of COX-2 mRNA, accompanied by a corresponding increase in PGE₂ biosynthesis. The anti-inflammatory steroid, dexamethasone (DEX) abolished the enhanced expression of COX-2 mRNA as well as PGE₂ formation induced by IL-1, PMA or the combination of IL-1 and PMA. The study indicates that the IL-1 induced PGE₂ formation is mediated by an enhanced gene expression of COX-2 in gingival fibroblasts suggesting that the enzyme COX-2 may play an important role in the regulation of prostanoid formation at inflammatory lesions in gingival tissue.     

HSV-1 promotes Ca-mediated APP phosphorylation and Aβ accumulation in rat cortical neurons

Epidemiological and experimental findings suggest that chronic infection with Herpes simplex virus type 1 (HSV-1) may be a risk factor for Alzheimer's disease (AD), but the molecular mechanisms underlying this association have not been fully identified. We investigated the effects of HSV-1 on excitability and intracellular calcium signaling in rat cortical neurons and the impact of these effects on amyloid precursor protein (APP) processing and the production of amyloid-β peptide (Aβ). Membrane depolarization triggering firing rate increases was observed shortly after neurons were challenged with HSV-1 and was still evident 12 hours postinfection. These effects depended on persistent sodium current activation and potassium current inhibition. The virally induced hyperexcitability triggered intracellular Ca²⁺ signals that significantly increased intraneuronal Ca²⁺ levels. It also enhanced activity- and Ca²⁺-dependent APP phosphorylation and intracellular accumulation of Aβ42. These findings indicate that HSV-1 causes functional changes in cortical neurons that promote APP processing and Aβ production, and they are compatible with the co-factorial role for HSV-1 in the pathogenesis of AD suggested by previous findings.     

Chronic exposure to endosulfan induces inflammation in murine colon via β-catenin expression and IL-6 production

Endosulfan (ENDO) is a widely used organochlorine (OC) pesticide and persistent organo-pollutant. Epidemiological studies have shown that high levels of OC exposure were related to colorectal cancer (CRC) incidence. The objectives of the present study were to evaluate histological changes in the colon, as well as in in situ expression of β-catenin and P-selectin, and serum levels of select pro-inflammatory cytokines in mice administered ENDO; there is a relationship between increased serum IL-6 and P-selectin levels in CRC patients and aberrant β-catenin signaling is important in initiation/maintenance of most CRCs. Mice were exposed to ENDO (at dose?50) orally once a week for up to 24 weeks, and monitored (inclusive) for a total of 42 weeks. The experiment was comprised of three groups, one that did not receive ENDO (olive oil vehicle), one administered 2?mg ENDO/kg/week and a positive control (for induction of CRC) given a weekly 20?mg 1,2-dimethylhydrazine (DMH)/kg injection. The results indicated that oral administration of ENDO provoked moderate inflammation starting at six weeks, and severe colonic inflammation with an appearance of dysplastic formations (aberrant crypts) in mice treated with ENDO (or DMH) for 12 weeks or longer. Serum IL-6 levels significantly increased starting at six weeks and rose to a peak of 15-fold higher than in controls at 42 weeks; TNFα levels likewise significantly increased, with a later peak (≈four-fold higher than controls) at 30–42 weeks. Immunohistochemical analysis of the colon also showed that expression of β-catenin and P-selectin increased with length of exposure to ENDO. Taken together, the results indicate that continued repeated oral exposure to ENDO induces increased expression of β-catenin and P-selectin, inflammation in the colon, and, ultimately, local tissue dysplasia.     

Silencing preBötzinger complex somatostatin-expressing neurons induces persistent apnea in awake rat

Delineating neurons that underlie complex behaviors is of fundamental interest. Using adeno-associated virus 2, we expressed the Drosophila allatostatin receptor in somatostatin (Sst)-expressing neurons in the preB?tzinger Complex (preB?tC). Rapid silencing of these neurons in awake rats induced a persistent apnea without any respiratory movements to rescue their breathing. We hypothesize that breathing requires preB?tC Sst neurons and that their sudden depression can lead to serious, even fatal, respiratory failure.     

Decreased expression of P-glycoprotein in interleukin-1β and interleukin-6 treated rat hepatocytes

OBJECTIVE AND DESIGN: As acute inflammation is known to cause a reduction in hepatic P-Glycoprotein (PGP) expression and activity in rats, we tested the hypothesis that the pro-inflammatory cytokines interleukin (IL-)1beta and IL-6 also mediate reductions in PGP. METHODS: Hepatocytes were incubated with 0-50 ng/ml of cytokine for 24-72 h. PGP/mdr expression was examined by immunodetection and quantitative RT-PCR analysis and PGP efflux activity was assayed. RESULTS: PGP protein was significantly reduced in cells treated for 3 days with IL-1beta and 24 h with IL-6 (p < 0.05), maximal effects occurring at 5 ng/ml for each cytokine. PGP activity was reduced in both IL-1beta and IL-6 treated cells (p < 0.05). mdr1 mRNA was decreased in cells treated with IL-6, but not IL-1beta. spgp and mdr2 were not affected. CONCLUSIONS: Our data indicate that IL-6 and IL-1beta have suppressive effects on the expression and activity of PGP in cultured hepatocytes, likely occurring through distinct mechanisms. These cytokines may have a potential role in PGP regulation during inflammatory responses.     

Discoordinate expression of IL-1α and IL-1β in interfacial membranes of failed joint prostheses

Bone resorption following either cemented or uncemented total hip replacement has been implicated as an important etiologic factor in aseptic loosening of prostheses, the most frequent cause of clinical failure. Interleukin-1 (IL-1), collagenase and prostaglandin E₂ are considered to play key roles in pathological bone resorption. We have measured the actual levels and quantified the genes coding for several cytokines [IL-1, IL-1, IL-4, IL-6, platelet-derived growth factors (PDGF), transforming growth factor- (TGF) and tumor necrosis factor- (TNF)] in interfacial membranes obtained from cemented or uncemented loosened joint replacements. IL-1, IL-6 and TNF were barely detectable in the interfacial membranes either at protein or mRNA levels, while IL-1 and TGF were found to be expressed at the highest levels in freshly isolated tissues. However, the expression of IL-1 increased 10–1000-fold either in isolated cells or explant cultures of interfacial membranes within 24 h. The expression of other cytokines, measured directly in tissue or cells, did not suggest a discoordinate expression of bone-resorbing cellular mediators.     

IL-33 enhances macrophage release of IL-1β and promotes pain and inflammation in gouty arthritis

Inflammation Research - To investigate the role of IL-33 in gouty arthritis. 174 Balb/c (wild-type) and 54 ST2?/? mice were used in this study. In vitro experiments were conducted in...     

Long-term IL-1β exposure causes subpopulation-dependent alterations in rat dorsal root ganglion neuron excitability

The effect of interleukin-1β (IL-1β) on the electrical properties of sensory neurons was assessed at levels and exposure times comparable to those found in animal models of neuropathic pain. Experiments involved whole cell current-clamp recordings from rat dorsal root ganglion (DRG) neurons in defined-medium, neuron-enriched cultures. Five- to six-day exposure to 100 pM IL-1β produced subpopulation-dependent effects on DRG neurons. These included an increase in the excitability of medium-diameter and small-diameter isolectin B(4) (IB(4))-positive neurons that was comparable to that found after peripheral nerve injury. By contrast, a reduction in excitability was observed in large-diameter neurons, while no effect was found in small-diameter IB(4)-negative neurons. Further characterization of changes in medium and small IB(4)-positive neurons revealed that some, but not all, effects of IL-1β were mediated through its receptor, IL-1RI. Although the acute actions of IL-1β on sensory neurons have been well studied and related to acute and/or inflammatory pain, the present study shows how sensory neurons respond to long-term cytokine exposure. Such effects are relevant to understanding processes that contribute to the onset of neuropathic pain.     

Human trophoblast-derived exosomal fibronectin induces pro-inflammatory IL-1β production by macrophages

PROBLEM Our previous studies demonstrated that trophoblast-derived exosomes induced synthesis and release of pro-inflammatory cytokines, including interleukin-1β (IL-1β) by macrophages. The objective of this study was to characterize the mechanism and receptors associated with this induction. METHOD OF STUDY Exosomes were isolated from Sw71 trophoblast-conditioned media by ultrafiltration and ultracentrifugation. Using macrophages isolated from normal donors, cytochalasin D was used to block exosome uptake. Induction of IL-1β mRNA was investigated by qRT-PCR, pro-IL-1β protein by western immunoblotting, and mature IL-1β release by ELISA. RGD peptides were used to block fibronectin binding by macrophage α5β1 integrin. RESULTS Uptake of exosomes by macrophages was completely blocked by pre-treatment with cytochalasin D. Although induction of some cytokines (such as C4A and CCL11) requires uptake, induction of IL-1β occurred without exosome internalization. Cytochalasin D treatment did not inhibit exosome-mediated induction of IL-1β mRNA, production of the pro-protein, or release of mature IL-1β. Blocking of fibronectin binding using RGD peptides demonstrated the abrogation of exosome-mediated IL-1β production. CONCLUSION Although trophoblast-derived exosomes have been demonstrated to induce IL-1β, this is the first demonstration of IL-1β induction by exosome-associated fibronectin. Based on this pro-inflammatory role of exosome-associated fibronectin, it may represent an important general immunoregulatory mechanism.     

Interleukin-1β in innate inflammation,autophagy and immunity

Although IL-1β is the master inflammatory cytokine in the IL-1 family, after more than ten years of continuous breeding, mice deficient in IL-1β exhibit no spontaneous disease. Therefore, one concludes that IL-1β is not needed for homeostasis. However, IL-1β-deficient mice are protected against local and systemic inflammation due to live infections, autoimmune processes, tumor metastasis and even chemical carcinogenesis. Based on a large number of preclinical studies, blocking IL-1β activity in humans with a broad spectrum of inflammatory conditions has reduced disease severity and for many, has lifted the burden of disease. Rare and common diseases are controlled by blocking IL-1β. Immunologically, IL-1β is a natural adjuvant for responses to antigen. Alone, IL-1β is not a growth factor for lymphocytes; rather in antigen activated immunocompetent cells, blocking IL-1 reduces IL-17 production. IL-1β markedly increases in the expansion of naive and memory CD4T cells in response to challenge with their cognate antigen. The response occurs when only specific CD4T cells respond to IL-1β and not to IL-6 or CD-28. A role for autophagy in production of IL-1β has emerged with deletion of the autophagy gene ATG16L1. Macrophages from ATG16L1-deficient mice produce higher levels of IL-1β after stimulation with TLR4 ligands via a mechanism of caspase-1 activation. The implications for increased IL-1β release in persons with defective autophagy may have clinical importance for disease.     

IL-36α expression is elevated in ulcerative colitis and promotes colonic inflammation

A role for the IL-36 family of cytokines has been identified in the pathogenesis of psoriasis. Although significant mechanistic overlap can exist between psoriasis and inflammatory bowel disease (IBD), to date there have been no reports investigating the IL-36 family in gastrointestinal inflammation. Here we demonstrate that expression levels of IL-36α are specifically elevated in the colonic mucosa of ulcerative colitis patients. This elevated expression is mirrored in the inflamed colonic mucosa of mice, wherein IL-36 receptor deficiency confirmed this pathway as a mediator of mucosal inflammation. Il36r−/− mice exhibited reduced disease severity in an acute DSS-induced model of colitis in association with decreased innate inflammatory cell infiltration to the colon lamina propria. Consistent with these data, infection with the enteropathogenic bacteria Citrobacter rodentium, resulted in reduced innate inflammatory cell recruitment and increased bacterial colonization in the colons of il36r−/− mice. Il36r−/− mice also exhibited altered T helper cell responses in this model, with enhanced Th17 and reduced Th1 responses, demonstrating that IL-36R signaling also regulates intestinal mucosal T-cell responses. These data identify a novel role for IL-36 signaling in colonic inflammation and indicate that the IL-36R pathway may represent a novel target for therapeutic intervention in IBD.     

Lipopolysaccharide-induced nigral inflammation leads to increased IL-1β tissue content and expression of astrocytic glial cell line-derived neurotrophic factor

Reactive gliosis and inflammatory change is a key component of nigral dopaminergic cell death in Parkinson's disease (PD). Astrocyte derived glial cell line-derived neurotrophic factor (GDNF) promotes the survival and growth of dopaminergic neurones and it protects against or reverses nigral degeneration induced by 6-OHDA and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in rodents and primates. But the effect of increased levels of pro-inflammatory cytokines on the release of GDNF is unknown. This study examined the relationship between release of tumour necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) and the expression of GDNF in rats following nigral lipopolysaccharide (LPS) administration. Acute nigral administration of LPS led to marked elevation of IL-1β but insignificant TNF-α tissue content and to a prominent expression of GDNF immunoreactivity in astrocytes but not microglia. The results suggest that inflammation is not only involved in neuronal loss but could promote neuronal survival through increased release of GDNF following up-regulation of IL-1β.     

Differential inflammasome expression and IL-1β secretion in monocyte-derived dendritic cells differentiated with IL-4 or IFN-α

Background

NLRP3-inflammasome activation was evaluated in monocyte-derived dendritic cells (DC) obtained through IL-4 (IL4-DC) or IFN-α (IFN-DC) protocols and pulsed with chemically inactivated HIV-1. Inflammasome’ genes expression and IL-1β secretion were compared in DC isolated from 15 healthy subjects (HC) and 10 HIV-1 infected individuals (HIV+).

Findings

Whether HIV was able to increased NLRP3-inflammasome genes expression and IL-1β secretion in IL4-DC from HC, the induction of inflammasome appeared significantly reduced in IFN-DC from HC, suggesting a different responsive state of IFN-DC compared to IL4-DC. No inflammasome activation was observed in IL4-DC as well as in IFN-DC derived from HIV?+?subjects, confirming previous findings on “unresponsive” state of DC derived from HIV?+?possibly due to chronic inflammatory state of these individuals.

Conclusions

Our results showed that IFN-α differently modulates inflammasome expression during monocytes-DC in vitro differentiation. These findings could be of interest considering the on-going research about DC manipulation and therapeutic strategies for HIV?+?involving DC-based immune-vaccines.

     

Short-term IL-1β blockade reduces monocyte CD11b integrin expression in an IL-8 dependent fashion in patients with type 1 diabetes

Objective

Interleukin 1-beta (IL-1β) is a major inflammatory cytokine. Blockade of the IL-1β pathway is therapeutically efficacious in type 2 diabetes, but the mechanistic effects on the immune system are incompletely understood.

Research design

We administered an IL-1 receptor antagonist, anakinra, to 7 type 1 diabetes patients in order to investigate the immunologic and metabolic effects of this drug. Mechanistic assays were performed before and after drug administration.

Results

A novel signature was observed, with reduced serum interleukin 8 (IL-8) levels and reduced CD11b integrin expression on monocytes associated with increased CXCR1 expression.

Conclusions

This set of linked phenotypes suggests that blockade of the IL-1β pathway results in the reduced ability of mononuclear cells to traffic to sites of inflammation. Mechanistic studies from large scale trials using IL-1 blockade in type 1 diabetes should focus on changes in monocyte trafficking and the IL-8 pathway.     

Distinct Contributions of Interleukin-1α (IL-1α) and IL-1β to Innate Immune Recognition of Pseudomonas aeruginosa in the Lung

The bacterial pathogen Pseudomonas aeruginosa causes acute infections associated with significant morbidity and mortality. P. aeruginosa elicits strong innate immune responses in immunocompetent hosts, and the resulting recruitment of neutrophils to the site of infection is necessary for bacterial clearance. P. aeruginosa lipopolysaccharide and flagellin are recognized by extracellular Toll-like receptors, but the most rapid responses to infection occur when cytosolic receptors sense flagellin or type 3 secretion system (T3SS) structural proteins. The subsequent activation of the NLRC4 inflammasome and caspase-1 generates an interleukin-1β (IL-1β) signal that is required for the rapid neutrophilic response. A T3SS effector, exotoxin U (ExoU), can inhibit activation of the NLRC4 inflammasome and caspase-1. Thus, our observation that IL-1 receptor (IL-1R)-mediated signals were still required to initiate a response to ExoU-producing bacteria was unexpected. As both IL-1α and IL-1β signal via the IL-1R, we examined immune responses in mice lacking either of these cytokines. IL-1β-deficient mice responded to ExoU-producing P. aeruginosa bacteria similarly to wild-type animals; however, IL-1α-deficient mice had an attenuated immune response. The situation was reversed following infections by ExoU-negative bacteria: here, IL-1α was dispensable for neutrophil recruitment, while IL-1β was required. IL-1α secretion by macrophages infected with ExoU-producing P. aeruginosa isolates was independent of both caspase-1 and caspase-11. This study documents distinct roles for IL-1α and IL-1β in the response to P. aeruginosa infection as a function of the T3SS effectors produced by the infecting strain. The redundancy of these two cytokines nonetheless allows the infected host to mount a response to ExoU-positive and -negative bacterial isolates.     

Effect of peptide aldehydes with IL-1β converting enzyme inhibitory properties on IL-1α and IL-1β production in vitro

Tripeptide and pentapeptide aldehydes as substrate-base inhibitors of cysteine proteases were designed in our laboratory for the inhibition of interleukin-lβ converting enzyme (ICE), a recently described cysteine protease responsible for the processing of IL-1β. The biological effectivity of the peptide aldehydes was studied in THP-1 cells and human whole blood. The released and cell-associated IL-1α and IL-1β levels were determined by ELISA from the supernatants and cell lysates, respectively. The total IL-1 like bioactivity was assayed by the D₁₀G₄₁ cell proliferation method. The tripeptide aldehyde (Z-Val-His-Asp-H) and pentapeptide aldehyde (Eoc-Ala-Tyr-Val-Ala-Asp-H) significantly reduced IL-1β levels in the supernatants in relatively high concentrations (10–100 μM), but the IL-1α release was unaffected by these peptides. However, a considerable decrease in the cell-associated IL-1β and IL-1α levels was observed. N-terminal extension of the tripeptide aldehyde yielded even more potent inhibitors. Amino acid substitution at the P₂ position did not cause considerable changes in the inhibitory activity. The peptide aldehydes suppressed the IL-1β production in a reversible manner, whereas dexamethasone, a glucocorticoid, had a prolonged inhibitory effect. The inhibitory effect of these peptides and that of dexamethasone appeared to be additive. These findings indicate that these peptide aldehydes might be used as IL-β inhibitory agents in experimental models in which IL-1β is a key mediator or ICE is implicated.