Production Of Multiple Pro-inflammatory Cytokines Research Articles

Lumbricus Extract Prevents LPS-Induced Inflammatory Activation of BV2 Microglia and Glutamate-Induced Hippocampal HT22 Cell Death by Suppressing MAPK/NF-κB/NLRP3 Signaling and Oxidative Stress.

Microglia-induced inflammatory signaling and neuronal oxidative stress are mutually reinforcing processes central to the pathogenesis of neurodegenerative diseases. Recent studies have shown that extracts of dried Pheretima aspergillum (Lumbricus) can inhibit tissue fibrosis, mitochondrial damage, and asthma. However, the effects of Lumbricus extracts on neuroinflammation and neuronal damage have not been previously studied. Therefore, to evaluate the therapeutic potential of Lumbricus extract for neurodegenerative diseases, the current study assessed the extract's anti-inflammatory and antioxidant activities in BV2 microglial cultures stimulated with lipopolysaccharide (LPS) along with its neuroprotective efficacy in mouse hippocampal HT22 cell cultures treated with excess glutamate. Lumbricus extract dose-dependently inhibited the LPS-induced production of multiple proinflammatory cytokines (tumor necrosis factor-α, interleukin (IL)-6, and IL-1β) and reversed the upregulation of proinflammatory enzymes (inducible nitric oxide synthase and cyclooxygenase-2). Lumbricus also activated the antioxidative nuclear factor erythroid 2-relayed factor 2/heme oxygenase-1 pathway and inhibited LPS-induced activation of the nuclear factor-κB/mitogen-activated protein kinases/NOD-like receptor family pyrin domain containing 3 inflammatory pathway. In addition, Lumbricus extract suppressed the glutamate-induced necrotic and apoptotic death of HT22 cells, effects associated with upregulated expression of antiapoptotic proteins, downregulation of pro-apoptotic proteins, and reduced accumulation of reactive oxygen species. Chromatography revealed that the Lumbricus extract contained uracil, hypoxanthine, uridine, xanthine, adenosine, inosine, and guanosine. Its effects against microglial activation and excitotoxic neuronal death reported herein support the therapeutic potential of Lumbricus for neurodegenerative diseases.

Pharmacological inhibition of IRAK4 kinase activity does not prevent cachexia in mice with pancreatic cancer

AbstractBackgroundInflammation is a hallmark of cachexia; however, effective anti‐inflammatory treatments have not yet been identified. Interleukin‐1 receptor‐associated kinase 4 (IRAK4) is a key signalling node linking interleukin‐1 receptor (IL‐1R) and toll‐like receptor (TLR) activation to the production of multiple proinflammatory cytokines that are elevated in cancer cachexia. The purpose of this work is to evaluate whether pharmacological inhibition of IRAK4 kinase activity with PF‐06426779 could prevent cachexia using a model of pancreatic cancer. The effect of appetite stimulation via the ghrelin receptor agonist anamorelin was also examined as a benchmark of clinically validated mechanisms.MethodsFemale C57Bl/6J mice were given an intraperitoneal injection of KrasG12D; p53R172H; Pdx1‐Cre (KPC) pancreatic tumour cells. PF‐06426779 or anamorelin treatment was initiated at the onset of anorexia. Body weight and food intake were measured throughout the study. Body composition, muscle function (force), and physical activity (treadmill running endurance) were assessed at the end of the study.ResultsChronic treatment with PF‐06426779, at doses covering in vitro IC50 and IC90 at Cmin, did not increase body weight, food intake, and muscle function in the KPC tumour model. In contrast, anamorelin (vs. vehicle) increased food intake (P < 0.01), hindlimb skeletal muscle mass (P < 0.01), and muscle strength (P < 0.05); however, treadmill running endurance was not increased.ConclusionsThese data suggest that inhibition of IRAK4 kinase activity is not sufficient to treat cachexia, at least in pancreatic cancer, and exploration of alternative anti‐inflammatory strategies that increase appetite is required.

Recombinant SARS-CoV-2 Spike Protein Stimulates Secretion of Chymase, Tryptase, and IL-1β from Human Mast Cells, Augmented by IL-33.

SARS-CoV-2 infects cells via its spike (S) protein binding to its surface receptor angiotensin-converting enzyme 2 (ACE2) and results in the production of multiple proinflammatory cytokines, especially in the lungs, leading to what is known as COVID-19. However, the cell source and the mechanism of secretion of such cytokines have not been adequately characterized. In this study, we used human cultured mast cells that are plentiful in the lungs and showed that recombinant SARS-CoV-2 full-length S protein (1-10 ng/mL), but not its receptor-binding domain (RBD), stimulates the secretion of the proinflammatory cytokine interleukin-1β (IL-1β) as well as the proteolytic enzymes chymase and tryptase. The secretion of IL-1β, chymase, and tryptase is augmented by the co-administration of interleukin-33 (IL-33) (30 ng/mL). This effect is mediated via toll-like receptor 4 (TLR4) for IL-1β and via ACE2 for chymase and tryptase. These results provide evidence that the SARS-CoV-2 S protein contributes to inflammation by stimulating mast cells through different receptors and could lead to new targeted treatment approaches.

Ameliorating effects of berberine on sepsis-associated lung inflammation induced by lipopolysaccharide: molecular mechanisms and preclinical evidence.

As a life-threatening disorder, sepsis-associated lung injury is a dysregulated inflammatory response to microbial infection, characterized by the infiltration of inflammatory cells into lung tissues and excessive production of pro-inflammatory mediators. Therefore, immunomodulatory/anti-inflammatory agents are a potential treatment for sepsis-associated lung injury. Berberine, one of the well-studied medicinal plant-derived compounds, has shown promising anti-inflammatory potential in inflammatory conditions, through modulating excessive immune responses induced by various immune cells. A systematic literature search in electronic databases indicated several publications that studied the effect of berberine on lipopolysaccharide (LPS)-induced sepsis in preclinical investigations. The current review article aims to provide evidence on the effects of berberine against LPS-induced acute lung injury (ALI), together with underlying molecular mechanisms. The findings reveal that berberine through inhibiting the excessive production of multiple pro-inflammatory cytokines, suppressing the infiltration of immune cells into lung tissues, as well as preventing pulmonary edema and coagulation, can relieve pulmonary histopathological changes from LPS-mediated inflammation, thereby attenuating sepsis-associated lung injury and lethality in the experimental models. In conclusion, berberine shows great potential as a preventing and therapeutic agent for sepsis-associated lung injury, however, further proof-of-concept studies and clinical investigations are warranted for translating these preclinical findings into clinical practices.

Multi-target regulation of pro-inflammatory cytokine production by transcription factor Blimp-1

ObjectiveCytokine storm syndrome is a fatal condition related to infectious and autoimmune diseases. Here, we aim to investigate the regulatory mechanisms of Blimp-1 on multiple cytokine production.MethodsThe Blimp1 shRNA was transfected into RAW264.7 macrophages, followed by Toll-like receptor (TLR) ligand stimulation. The mRNA and protein levels of cytokines were detected by real-time PCR and flow cytometric bead array. The nuclear translocation of AP-1 and NF-κB p65 was measured by immunofluorescence staining. The transcriptional activity was detected by luciferase reporter assay with 5 × NF-κB reporter or with IL6 promoter reporter.ResultsBlimp-1 significantly inhibited the expression and secretion of IL-1β, IL-6, and IL-18 in macrophages during stimulation with a variety of TLR ligands. The immunofluorescence staining results showed that Blimp-1 strictly controlled the nuclear translocation of NF-κB p65 in LPS-challenged macrophages. Furthermore, Blimp-1 directly inhibited the transcriptional activity of NF-κB and the transcription of IL6 gene.ConclusionBlimp-1 represses the production of multiple pro-inflammatory cytokines by directly binding the genomic region and restricting the nuclear translocation and transcriptional activity of NF-κB. This finding may provide potential therapeutic strategies for the cytokine storm-related diseases.

High-density lipoprotein reduces microglia activation and protects against experimental autoimmune encephalomyelitis in mice

Studies have shown that high-density lipoprotein (HDL) is a powerful anti-atherosclerosis factor in vivo and in vitro, with anti-inflammatory effects, and it also plays an important role in the immune system and central nervous system (CNS). In this study, the BV2 microglia inflammation model and experimental autoimmune encephalomyelitis animal model were used to investigate the potential mechanism of HDL in multiple sclerosis. Our results show that HDL inhibits the activation of BV2 microglia in a model of BV2 microglia inflammation and were validated with primary microglia. HDL can down-regulate the expression of TNF-α, IL-6, iNOS and NO. Western blot results showed that HDL could reduce the expression levels of TLR4, CD14, MyD88 and NF-κB p65 LPS-induced microglia. In a mouse model of experimental autoimmune encephalomyelitis, hematoxylin-eosin (HE) staining showed decreased infiltration of inflammatory cells in brain and spinal cord tissues, and Luxol Fast Blue (LFB) staining showed significant improvement in spinal cord demyelination. We found that HDL reduced spinal cord and brain inflammation after EAE induction, inhibited the infiltration of CD68 and Iba-1 positive inflammatory cells, and reduced the production of multiple pro-inflammatory cytokines, including pro-inflammatory cytokines TNF-α, IL-6, and IL-1β. Western blot showed that EAE mice HDL inhibited the activation of ERK1/2 and JNK in MAPK pathway and p-IκBα and P65 in NF-κB pathway. Taken together, our study suggests that HDL may influence microglia activation and inflammatory response in mice by regulating inflammatory signaling pathways, improving induction of experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis, and provides further insights into HDL therapy for multiple sclerosis.

Synthesis and pharmacological evaluation of choroquine derivatives bearing long aminated side chains as antivirus and anti-inflammatory agents

Starting from the antimalarial drugs chloroquine and hydroxychloroquine, we conducted a structural optimization on the side chain of chloroquine by introducing amino substituted longer chains thus leading to a series of novel aminochloroquine derivatives. Anti-infectious effects against SARS-Cov2 spike glycoprotein as well as immunosuppressive and anti-inflammatory activities of the new compounds were evaluated. Distinguished immunosuppressive activities on the responses of T cell, B cell and macrophages upon mitogen and pathogenic signaling were manifested. Compounds 9–11 displayed the most promising inhibitory effects both on cellular proliferation and on the production of multiple pro-inflammatory cytokines, including IL-17, IFN-γ, IL-6, IL-1β and TNF-α, which might be insightful in the pursuit of treatment for immune disorders and inflammatory diseases.

Anti-inflammatory activity of soluble chito-oligosaccharides (CHOS) on VitD3-induced human THP-1 monocytes.

Chito-oligosaccharides (CHOS) are oligomers of D-glucosamine and N-acetyl-glucosamine. Anti-inflammatory activities of a wide variety of CHOS mixtures have previously been reported, mainly based on studies with mouse models and murine macrophages. Since the mouse and human immune systems are quite different, gaining insight into the activity of CHOS on human cell lines, using well-characterized CHOS mixtures, is of considerable interest. Bacillus subtilis chitosanase (BsCsn46A) can efficiently convert chitosan to mixtures of water soluble low molecular weight CHOS. Here, the anti-inflammatory activity of a properly characterized CHOS mixture was studied, using human THP-1 cells that were differentiated to mature monocytes using vitamin D3. Addition of CHOS reduced the production of multiple pro-inflammatory cytokines associated with bacterial lipopolyssacharide (LPS)-stimulated inflammation, in a dose-dependent manner and without affecting cell viability. Interestingly, only minimal effects of CHOS were observed in similar experiments with phorbol 12-myristate 13-acetate- (PMA-) differentiated, macrophage-like, THP-1 cells. Altogether, in addition to showing promising biological effects of well-characterized low molecular weight soluble CHOS in a human system, the present study also points at Vitamin D3-stimulated THP-1 cells as a favorable system for assessing the anti-inflammatory activity of bioactive compounds.

Nanomedicine therapies modulating Macrophage Dysfunction: a potential strategy to attenuate Cytokine Storms in severe infections.

Cytokine storms, defined by the dysregulated and excessive production of multiple pro-inflammatory cytokines, are closely associated with the pathology and mortality of several infectious diseases, including coronavirus disease 2019 (COVID-19). Effective therapies are urgently needed to block the development of cytokine storms to improve patient outcomes, but approaches that target individual cytokines may have limited effect due to the number of cytokines involved in this process. Dysfunctional macrophages appear to play an essential role in cytokine storm development, and therapeutic interventions that target these cells may be a more feasible approach than targeting specific cytokines. Nanomedicine-based therapeutics that target macrophages have recently been shown to reduce cytokine production in animal models of diseases that are associated with excessive proinflammatory responses. In this mini-review, we summarize important studies and discuss how macrophage-targeted nanomedicines can be employed to attenuate cytokine storms and their associated pathological effects to improve outcomes in patients with severe infections or other conditions associated with excessive pro-inflammatory responses. We also discuss engineering approaches that can improve nanocarriers targeting efficiency to macrophages, and key issues should be considered before initiating such studies.

Abstract 3192: Activation of p53 in the tumor microenvironment by MDM2 inhibitor APG-115 synergizes with PD-1 blockade independently of p53 status of tumor cells

Abstract Blockade of the checkpoint inhibitor programmed death 1 (PD-1) has gained big success in cancer therapy. However, the response rate of anti-PD1 agents remains low. Molecularly targeted agents offer selectivity and high tumor response rates, but patients develop resistance to these drugs inevitably. Combinations of targeted agents and immunotherapy provide new opportunities to improve cancer treatments. Recent studies found that p53 activation in the myeloid linage suppressed alternative (M2) macrophage polarization and attenuates tumor development and invasion, leading to the hypothesis that p53 activation may further augment antitumor immunity elicited by anti-PD-1 therapy. APG-115 is an orally active, selective, small molecule inhibitor of the MDM2-p53 protein-protein interaction. APG-115 acts as an antitumor agent by activating the p53 tumor suppressor in p53 wild-type tumors. However, its role in regulating immune responses remained unknown. In this study, we investigated the role of APG-115 in immune modulation both in vitro and in vivo. Enhanced antitumor activity was first demonstrated in p53 wild-type MH-22A, p53 mutant MC38, and p53 knockout (p53-/-) MH-22A syngeneic tumor models after the combination treatment of APG-115 and anti-PD-1 antibody. Despite differential changes in tumor-infiltrating leukocytes, including an increase in cytotoxic CD8+ T cells in the p53 wild-type tumors and an increase in proinflammatory M1 macrophages in the p53 mutant tumors, the combination treatment consistently reduced immunosuppressive M2 macrophages in the tumor microenvironment regardless of p53 status of tumor cells. In addition, in vitro, the treatment of bone marrow-derived macrophages with APG-115 resulted in activation of p53 and p21 gene expression, as well as a decrease in M2 macrophages population and reduction of c-MYC and M2-related gene expression. Moreover, enhanced M1 macrophage polarization in the spleen was also observed in naïve mice treated with APG-115. Furthermore, APG-115 increased production of multiple proinflammatory cytokines, including IFN-γ, TNF-α, IL-2 and IL-6, in stimulated T cells. Collectively, for the first time, our findings suggest that p53 activation by a pharmacological MDM2 inhibitor enables reversal of immunosuppressive tumor microenvironment and enhance antitumor immunity independently of p53 status of tumors. Specifically, in complementary to PD-1 blockade that predominantly activates cytotoxic CD8+ T cell populations, APG-115 primarily targets tumor-associated macrophages. Collectively, our data provide a rationale for applying the combination of APG-115 plus PD-1 blockade to a broader patient population with p53 mutant tumors. Accordingly, a clinical trial of APG-115 in combination with pembrolizumab in metastatic melanoma patients regardless of p53 status has been initiated in the USA. Citation Format: Douglas D. Fang, Qiuqiong Tang, Yanhui Kong, Qixin Wang, Jiaxing Gu, Xu Fang, Peng Zou, Tao Rong, Jingwen Wang, Dajun Yang, Yifan Zhai. Activation of p53 in the tumor microenvironment by MDM2 inhibitor APG-115 synergizes with PD-1 blockade independently of p53 status of tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3192.

IGF2BP1 promotes LPS-induced NFκB activation and pro-inflammatory cytokines production in human macrophages and monocytes

BackgroundLipopolysaccharide (LPS)-induced macrophage/monocyte activation and pro-inflammatory cytokines production are important mediators for periodontitis progression. The current study tested the potential role of insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) in the process. MethodsTHP-1 human macrophages and primary human peripheral blood mononuclear cells (PBMCs) were treated with LPS. mRNA and protein expression of IGF2BP1 were tested by qPCR and Western blotting assay. IGF2BP1 expression was altered by shRNAs or CRISPR/Cas-9 gene editing methods. LPS-induced cytokine production was tested by ELISA assay. Cytokine mRNA expression was tested by the quantitative real-time reverse transcriptase polymerase chain reaction (qPCR) assay. ResultsIn THP-1 human macrophages and PBMCs, treatment with LPS induced mRNA and protein expression of IGF2BP1. IGF2BP1 silencing (by targeted shRNAs) or CRISPR/Cas-9 knockout largely inhibited LPS-induced production of multiple pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6. Conversely, forced over-expression of IGF2BP1 facilitated LPS-induced pro-inflammatory cytokines production in THP-1 cells. For the mechanism study, we show that IGF2BP1 co-immunoprecipitated with p65-p52 nuclear factor kappa B (NFκB) complex in nuclei of LPS-treated THP-1 cells. Significantly, LPS-induced p65-p52 nuclear translocation and NFκB activation were inhibited by IGF2BP1 silencing or CRISPR/Cas-9 knockout. ConclusionIGF2BP1 promotes LPS-induced NFκB signalling and transcriptional activation in human macrophages and monocytes.

The miR-183/96/182 Cluster Regulates Macrophage Functions in Response to Pseudomonas aeruginosa

Macrophages (Mϕ) are an important component of the innate immune system; they play critical roles in the first line of defense to pathogen invasion and modulate adaptive immunity. MicroRNAs (miRNAs) are a newly recognized, important level of gene expression regulation. However, their roles in the regulation of Mϕ and the immune system are still not fully understood. In this report, we provide evidence that the conserved miR-183/96/182 cluster (miR-183/96/182) modulates Mϕ function in their production of reactive nitrogen (RNS) and oxygen species (ROS) and their inflammatory response to Pseudomonas aeruginosa (PA) infection and/or lipopolysaccharide (LPS) treatment. We show that knockdown of miR-183/96/182 results in decreased production of multiple proinflammatory cytokines in response to PA or LPS treatment in Mϕ-like Raw264.7 cells. Consistently, peritoneal Mϕ from miR-183/96/182-knockout versus wild-type mice are less responsive to PA or LPS, although their basal levels of proinflammatory cytokines are increased. In addition, overexpression of miR-183/96/182 results in decreased production of nitrite and ROS in Raw264.7 cells. We also provide evidence that DAP12 and Nox2 are downstream target genes of miR-183/96/182. These data suggest that miR-183/96/182 imposes global regulation on various aspects of Mϕ function through different downstream target genes.

4-Octyl Itaconate Activates Nrf2 Signaling to Inhibit Pro-Inflammatory Cytokine Production in Peripheral Blood Mononuclear Cells of Systemic Lupus Erythematosus Patients

Background/Aims: Increased production of multiple pro-inflammatory cytokines, including tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6, plays an essential pathogenic role in the progression of systemic lupus erythematosus (SLE). Recent studies have characterized itaconate as a novel and potent nuclear-factor-E2-related factor 2 (Nrf2) activator that activates Nrf2 signaling by alkylating cysteine residues on Keap1 (Kelch-like ECH-associated protein 1). Methods: THP-1 human macrophages and peripheral blood mononuclear cells (PBMCs) of SLE patients were treated with 4-octyl itaconate (OI). Nrf2 signaling activation was tested by qPCR assay and western blotting. mRNA expression and the production of multiple pro-inflammatory cytokines were tested by qPCR and enzyme-linked immunosorbent assays, respectively. Nuclear factor (NF)-κB activation was tested by the p65 DNA-binding assay. Results: We demonstrated that OI, the cell-permeable derivative of itaconate, induced Keap1-Nrf2 dissociation, Nrf2 protein accumulation, and nuclear translocation, which enabled the transcription and expression of multiple Nrf2-dependentantioxidant enzymes (heme oxygenase-1, NAD(P)H:quinone oxidoreductase 1, and glutamate-cysteine ligase modifier subunit) in THP-1 human macrophages. OI also induced significant Nrf2 activation in SLE patient-derived PBMCs. OI pretreatment inhibited mRNA expression and the production of multiple pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) in SLE patient-derived PBMCs and lipopolysaccharide (LPS)-activated THP-1 cells. OI potently inhibited NF-κB activation in SLE patient-derived PBMCs and LPS-activated THP-1 cells. Importantly, Nrf2 silencing (by targeted short hairpin RNA) or knockout (by CRISPR/Cas9 gene-editing method) almost abolished OI-induced anti-oxidant and anti-inflammatory actions in SLE patient-derived PBMCs and LPS-activated THP-1 cells. Conclusion: OI activates Nrf2 signaling to inhibit the production of pro-inflammatory cytokines in human macrophages and SLE patient-derived PBMCs. OI and itaconate could have important therapeutic value for the treatment of SLE.

Abstract 2690: MK2 pathway blockade inhibits inflammatory cytokine production and colorectal cancer growth and invasion

Abstract Introduction: Colorectal cancer is the third most common malignancy diagnosed for both men and women in the United States of America. High levels of inflammatory cytokines in colorectal tumors cause increased growth and invasion and carry a higher risk of metastasis. Identifying new targets to control inflammation is important for developing improved treatment approaches. Mitogen-activated protein kinase-activated protein kinase 2 (MK2) is a regulator of pro-inflammatory cytokines that may promote colorectal tumor progression. MK2 signaling is known to induce IL-1, IL-6, and TNF-α production. These pro-inflammatory cytokines are associated with CRC development, invasion, and metastasis. We hypothesized that the MK2 pathway could be an important component of CRC growth, invasion and tumor regrowth. Methods: To investigate this pathway, CT26 CRC cells were examined. Cells were flank-injected into Balb/c mice with and without MK2 inhibitor treatment. At day 19 after tumor injection, tumors were harvested and measured using calipers. Cytokines were quantitated by multiplex bead array in organ culture supernatants and in supernatants from tumor cells plated in fibronectin coated wells. In culture, tumor cell invasion was measured in scratch wound assays containing matrigel. Results: Treating CT26 cells with MK2 inhibitors markedly reduced tumor growth in mice by a mean of 60% compared to vehicle control treated cells. Inflammatory cytokines were also dramatically decreased with MK2 inhibition compared to controls by up to 80%. Cytokines affected included both known MK2 downstream cytokines (IL-1, IL-6, and TNF-α) and also chemokines such as MIP-1α and MCP-1 in both supernatants from cultured tumor cells and in organ culture supernatants. MK2 inhibition also decreased invasion of tumor cells in a cytokine dependent manner. Conclusions: The MK2 pathway regulates production of multiple pro-inflammatory cytokines in colorectal tumors, including several previously unreported chemokines. Inhibition of this pathway markedly decreases tumor growth and invasion. Thus, MK2 may be a promising tumor target to prevent colorectal cancer progression. Citation Format: Anita L. Ray, Amanda S. Peretti, Wade Johnson, Gregory Gan, Ellen J. Beswick. MK2 pathway blockade inhibits inflammatory cytokine production and colorectal cancer growth and invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2690. doi:10.1158/1538-7445.AM2017-2690

An aberrant immune response to an ocular commensal results in disease in a mouse model of Muckle-Wells Syndrome

Abstract Appropriate regulation of host immunity by commensal bacteria at mucosal tissues, including the ocular surface, can prevent infection and limit disease. Our recent data (St. Leger et al, this conference) suggest that Corynebacterium mastitidis (=C. mast), an ocular commensal, elicits production of protective IL-17 from conjunctival gdT cells. What is not known is whether this relationship, when dysregulated, can lead to pathology and disease. In this study, we used a mouse model of Muckle-Wells Syndrome (MWS), which is a human autoinflammatory disease that results in spontaneous inflammation of the conjunctiva, skin and joints. This is caused by a mutation in the NLRP3 gene (cias1) that leads to an overactive NLRP3 inflammasome and results in production of multiple proinflammatory cytokines, including, most prominently, IL-1b. Here, we show that ocular colonization with a commensal bacterium, C. mast, induces ocular inflammation in MWS mice, but not in WT controls. Dendritic cells from MWS mice produced increased amounts of IL-1b upon stimulation with C. mast lysates. In keeping with this, DCs harvested from MWS mice more efficiently activated gdT cells (in particular, Vγ4+ cells) in co-culture experiments, compared to DCs from WT mice. Our results suggest that the commensal C. mast can act as a pathobiont and trigger ocular inflammation in mice with an overactive NLRP3 inflammasome. Thus, an aberrant immune response to commensal microbes in humans with this mutation may be the cause of recurrent conjunctivitis in patients with MWS or similar autoinflammatory diseases.

Intranasal delivery of FSD-C10, a novel Rho kinase inhibitor, exhibits therapeutic potential in experimental autoimmune encephalomyelitis.

Viewing multiple sclerosis (MS) as both neuroinflammation and neurodegeneration has major implications for therapy, with neuroprotection and neurorepair needed in addition to controlling neuroinflammation in the central nervous system (CNS). While Fasudil, an inhibitor of Rho kinase (ROCK), is known to suppress experimental autoimmune encephalomyelitis (EAE), an animal model of MS, it relies on multiple, short-term injections, with a narrow safety window. In this study, we explored the therapeutic effect of a novel ROCK inhibitor FSD-C10, a Fasudil derivative, on EAE. An important advantage of this derivative is that it can be used via non-injection routes; intranasal delivery is the preferred route because of its efficient CNS delivery and the much lower dose compared with oral delivery. Our results showed that intranasal delivery of FSD-C10 effectively ameliorated the clinical severity of EAE and CNS inflammatory infiltration and promoted neuroprotection. FSD-C10 effectively induced CNS production of the immunoregulatory cytokine interleukin-10 and boosted expression of nerve growth factor and brain-derived neurotrophic factor proteins, while inhibiting activation of p-nuclear factor-κB/p65 on astrocytes and production of multiple pro-inflammatory cytokines. In addition, FSD-C10 treatment effectively induced CD4(+) CD25(+) , CD4(+) FOXP3(+) regulatory T cells. Together, our results demonstrate that intranasal delivery of the novel ROCK inhibitor FSD-C10 has therapeutic potential in EAE, through mechanisms that possibly involve both inhibiting CNS inflammation and promoting neuroprotection.

Abstract 3986: Protein-bound polysaccharide induces IL-1β via NLRP3 inflammasome activation.

Abstract Protein-bound polysaccharide-K (PSK) is a hot water extract from Trametes Versicolor mushroom. It is widely used in Asian countries for its immune enhancing and anti-cancer potential. We have recently reported that PSK activates TLR2 and has CD8 T cell- and NK cell- dependent anti-tumor activity. PSK stimulates the production of multiple pro-inflammatory cytokines, including IL-1β, the production of which requires both TLR and inflammasome activation. The current study was undertaken to investigate the mechanism by which PSK activates inflammasome and induces IL-1β. RT-PCR and Western blot analyses showed that PSK induces pro-IL-1β in a dose- and time- dependent manner. PSK activates caspase-1 and pre-incubation with z-VAD-FMK, a caspase inhibitor, abolishes PSK-induced IL-1β. Comparison of THP-1 cells and THP-1 cells deficient for NLRP3 demonstrated that PSK-induced IL-1β production requires NLRP3 inflammasome. PSK also up-regulates the expression of NLRP3 mRNA. Further experiments were performed to investigate whether potassium efflux, reactive oxygen species (ROS) generation, or lysosomal perturbation was involved in PSK-induced NLRP3 inflammasome activation. Results showed that CA-074-Me, a cathepsin B inhibitor, significantly decreased PSK-induced IL-1β, while the production of TNF-α was not affected. Phagocytosis or ROS generation was not required for PSK-induced NLRP3 inflammasome activation. Altogether, our results suggest that PSK-induced IL-1β production is a two step process. Step 1 requires TLR2-dependent induction of pro-IL-1β and upregulation of NLRP3 expression; Step 2 involves NLRP3 inflammasome activation in a cathepsin B-dependent manner. These results provide novel insights into the mechanisms of the immune stimulatory and anti-tumor effects of PSK. Citation Format: Yi Yang, Carol Inatsuka, Mary L. Disis, Hailing Lu. Protein-bound polysaccharide induces IL-1β via NLRP3 inflammasome activation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3986. doi:10.1158/1538-7445.AM2013-3986

Celecoxib decreases the production of cytokines by osteoarthritic cartilage and synovium without affecting cartilage matrix degradation

Purpose: Recently, we reported a coculture model with osteoarthritic cartilage and synovial tissue that is more representative for osteoarthritis (OA) and useful to study mechanisms of action of OA therapies. Celecoxib, a COX-2 inhibitor, is frequently used for the treatment of osteoarthritis. However, its effect on cartilage metabolism has been the subject of much debate. Moreover, it is unclear how celecoxib affects the levels of soluble mediators in the joint. In the current study we investigate the effect of celecoxib on cartilage matrix turnover and the secretion of soluble mediators by cartilage and synovium in a coculture of OA cartilage and synovial tissue. Methods: OA cartilage and OA synovium explants were cultured alone or in coculture for 21 days with the addition of celecoxib at 0.1, 1.0 and 10 μM. To study cartilage matrix turnover glycosaminoglycan (GAG) content, production and release were determined. ELISAs for IL-1α, IL-1ß, IL-4, IL-6, IL-8, IL-10, IL-13, OSM, MCP1, NGF, leptin, adiponectin and PGE2 were performed on conditioned medium of day 4. For statistical analysis, univariate analysis of variance was performed, with a randomized block design to correct for inter-donor variability. P values < 0.05 were considered significantly different. Results: Both osteoarthritic cartilage and synovial tissue produced PGE2 and celecoxib inhibited the production in a dose dependent manner (P < 0.05; Fig. 1a). Celecoxib, irrespective of the concentration used, did not show an effect on GAG content, release and production in the coculture (Fig. 1b). Celecoxib at a concentration of 10 μM decreased the production of IL-1α, IL-1ß, IL-6, IL-10, OSM and NGF (P < 0.05). No effect was seen on leptin, adiponectin, IL-8 and MCP1. IL-13 and IL-4 were not detected (Fig. 1c). Conclusion: This study shows that celecoxib decreased the production of multiple soluble mediators associated with inflammation and pain in OA. Although the production of multiple pro-inflammatory cytokines and PGE2 was decreased, no effect on cartilage matrix turnover was seen. The decreased production of mediators associated with pain in OA, such as NGF, IL-6 and PGE2 is in line with the effect on pain observed in patients treated with celecoxib.

Poly-Thymidine Oligonucleotides Mediate Activation of Murine Glial Cells Primarily Through TLR7, Not TLR8

The functional role of murine TLR8 in the inflammatory response of the central nervous system (CNS) remains unclear. Murine TLR8 does not appear to respond to human TLR7/8 agonists, due to a five amino acid deletion in the ectodomain. However, recent studies have suggested that murine TLR8 may be stimulated by alternate ligands, which include vaccinia virus DNA, phosphothioate oligodeoxynucleotides (ODNs) or the combination of phosphothioate poly-thymidine oligonucleotides (pT-ODNs) with TLR7/8 agonists. In the current study, we analyzed the ability of pT-ODNs to induce activation of murine glial cells in the presence or absence of TLR7/8 agonists. We found that TLR7/8 agonists induced the expression of glial cell activation markers and induced the production of multiple proinflammatory cytokines and chemokines in mixed glial cultures. In contrast, pT-ODNs alone induced only low level expression of two cytokines, CCL2 and CXCL10. The combination of pT-ODNs along with TLR7/8 agonists induced a synergistic response with substantially higher levels of proinflammatory cytokines and chemokines compared to CL075. This enhancement was not due to cellular uptake of the agonist, indicating that the pT-ODN enhancement of cytokine responses was due to effects on an intracellular process. Interestingly, this response was also not due to synergistic stimulation of both TLR7 and TLR8, as the loss of TLR7 abolished the activation of glial cells and cytokine production. Thus, pT-ODNs act in synergy with TLR7/8 agonists to induce strong TLR7-dependent cytokine production in glial cells, suggesting that the combination of pT-ODNs with TLR7 agonists may be a useful mechanism to induce pronounced glial activation in the CNS.

Pirfenidone Inhibits T-Cell Activation, Proliferation, Cytokine and Chemokine Production, and Host Alloresponses

We previously showed that pirfenidone, an anti-fibrotic agent, reduces lung allograft injury or rejection. In this study, we tested the hypothesis that pirfenidone has immune modulating activities and evaluated its effects on the function of T-cell subsets, which play important roles in allograft rejection. We first evaluated whether pirfenidone alters T-cell proliferation and cytokine release in response to T-cell receptor (TCR) activation, and whether pirfenidone alters regulatory T cells (CD4CD25) suppressive effects using an in vitro assay. Additionally, pirfenidone effects on alloantigen-induced T-cell proliferation in vivo were assessed by adoptive transfer of carboxyfluorescein diacetate succinimidyl ester-labeled T cells across a parent->F1 major histocompatibility complex mismatch, as well as using a murine heterotopic cardiac allograft model (BALB/c->C57BL/6). Pirfenidone was found to inhibit the responder frequency of TCR-stimulated CD4 cell total proliferation in vitro and in vivo, whereas both CD4 and CD8 proliferation index were reduced by pirfenidone. Additionally, pirfenidone inhibited TCR-induced production of multiple pro-inflammatory cytokines and chemokines. Interestingly, there was no change on transforming growth factor-beta production by purified T cells, and pirfenidone had no effect on the suppressive properties of naturally occurring regulatory T cells. Pirfenidone alone showed a small but significant (P<0.05) effect on the in vivo allogeneic response, whereas the combination of pirfenidone and low dose rapamycin had more remarkable effect in reducing the alloantigen response with prolonged graft survival. Pirfenidone may be an important new agent in transplantation, with particular relevance to combating chronic rejection by inhibiting both fibroproliferative and alloimmune responses.