RESUMO
BACKGROUND: Cancer and its therapies can impact fertility in various ways, and therefore a growing number of cancer survivors face fertility as a significant concern. The cytotoxic alkylating agent cyclophosphamide (CP) is commonly used as an antineoplastic agent; unfortunately, its use is significantly associated with male infertility and damage to the reproductive system. AIM: The present study aimed to assess the possible beneficial effects of Irbesartan (IRB) in a rat model of CP-induced testicular toxicity. MAIN METHODS: The effects of treatment were assessed by measuring peroxisome proliferator-activated receptor gamma (PPAR-γ) expression via qRT-PCR, the immunohistochemical (IHC) assessment of apoptotic markers, NOD-like receptor protein 3 (NLRP3), and nuclear factor-κB (NF-κB), determination of the count and viability of epididymal sperm, oxidative stress markers via biochemical analysis, serum testosterone, caspase-1, and interleukin-18 (IL-18) levels via ELISA, histopathological assessment, and fibrosis by Masson's trichrome (MT) stain. KEY FINDINGS: There was a significant increase in malondialdehyde (MDA), caspase-1, and IL-18 contents, NF-κB, NLRP3, Bcl-2-associated X protein (Bax), caspase-3, and MT staining in testicular tissue after CP administration compared to the normal control group. Whereas reduced glutathione (GSH), superoxide dismutase (SOD), PPAR-γ expression, B-cell lymphoma-2 (Bcl-2) staining, serum testosterone, and the count and viability of epididymal sperm were decreased compared to the normal control group. The IRB treatment has reversed CP-induced testicular toxicity. SIGNIFICANCE: It is possible to conclude that IRB revealed a significant testicular protective effect against CP via antioxidant, anti-apoptotic, and anti-inflammatory effects.
Assuntos
Ciclofosfamida/efeitos adversos , Interleucina-18/biossíntese , Irbesartana/farmacologia , NF-kappa B/biossíntese , Proteína 3 que Contém Domínio de Pirina da Família NLR/biossíntese , PPAR gama/biossíntese , Transdução de Sinais/efeitos dos fármacos , Doenças Testiculares , Regulação para Cima/efeitos dos fármacos , Animais , Ciclofosfamida/farmacologia , Infertilidade Masculina/induzido quimicamente , Infertilidade Masculina/tratamento farmacológico , Infertilidade Masculina/metabolismo , Infertilidade Masculina/patologia , Masculino , Ratos , Doenças Testiculares/induzido quimicamente , Doenças Testiculares/tratamento farmacológico , Doenças Testiculares/metabolismo , Doenças Testiculares/patologiaRESUMO
BACKGROUND: N-Acetylcysteine (NAC) had exerted antioxidation and anti-inflammation effects on chronic obstructive pulmonary disease (COPD) patients. However, its effect in regulating interleukin- (IL-) 18 was not fully understood. This study was designed to evaluate the specific mechanism of NAC regulating IL-18. MATERIALS AND METHODS: A total of 112 COPD patients and 103 health individuals were recruited in the study. Cytokine level in patients' serum was measured by enzyme-linked immunosorbent assay (ELISA). A COPD mouse model was established by administration of lipopolysaccharide (LPS) and cigarette smoke. The expression of cytokines was measured by ELISA and flow cytometry. Inflammasome-related protein was measured by Western blot. RESULT: NAC could effectively improve the immune status of COPD patients as well as the COPD mouse model by downregulating proinflammation and inflammation cytokines including IL-1ß, interferon- (IFN-) γ, tumor necrosis factor- (TNF-) α, and IL-18. It also had the capability to suppress synthesis of IL-18 in macrophage to inhibit the secretion of IFN-γ from natural killer (NK) cells through influencing the inflammasome-related protein in macrophages. CONCLUSION: NAC could effectively inhibit the production of IL-18 by suppressing NLRP3 expression in macrophages to reduce the production of IFN-γ in NK cells.
Assuntos
Acetilcisteína/farmacologia , Interferon gama/biossíntese , Interleucina-18/biossíntese , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidores , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Doença Pulmonar Obstrutiva Crônica/imunologia , Idoso , Animais , Estudos de Casos e Controles , Biologia Computacional , Citocinas/sangue , Modelos Animais de Doenças , Feminino , Humanos , Interleucina-18/antagonistas & inibidores , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Pulmão/patologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Doença Pulmonar Obstrutiva Crônica/patologiaRESUMO
OBJECTIVE: To observe the protective effect of AC-YVAD-CMK on sepsis-induced acute kidney injury in mice and to explore its possible mechanisms primarily. METHODS: Eighteen male C57BL/6 mice were randomly divided into sham-operated group (Control), cecal ligation and puncture group (CLP), and CLP model treated with AC-YVAD-CMK group (AC-YVAD-CMK) (n = 6 in each group). Mice were sacrificed at 24 h after operation, and blood and kidney tissue samples were collected for analyses. Histologic changes were determined microscopically following HE staining. The expression of Ly-6B and CD68 was investigated using immunohistochemistry. Serum concentrations of creatinine (sCR) and blood urea nitrogen (BUN) were measured. Serum levels of interleukin-1ß (IL-1ß), interleukin-18 (IL-18), TNF-α, and interleukin-6 (IL-6) were determined by ELISA. The expressions of Caspas-1, NLRP-1, IL-1ß, and IL-18 in renal tissues were investigated using Western blot. Immunofluorescence staining was used to detect the expression of GSDMD protein in renal tissues. RESULTS: AC-YVAD-CMK treatment significantly alleviates sepsis-induced acute kidney injury, with decreased histological injury in renal tissues, suppresses the accumulation of neutrophils and macrophages in renal tissues, and decreased sCR and BUN level (P < 0.05). Attenuation of sepsis-induced acute kidney injury was due to the prohibited production of inflammatory cytokines and decrease expression of Caspas-1, NLRP-1, IL-1ß, and IL-18 in renal tissues. In addition, AC-YVAD-CMK treatment significantly reduced the expression of GSDMD in renal tissues compared to those observed in controls (P < 0.05). CONCLUSIONS: We demonstrated a marked renoprotective effect of caspase-1-inhibitor AC-YVAD-CMK in a rat model of sepsis by inhibition of pyroptosis.
Assuntos
Injúria Renal Aguda/tratamento farmacológico , Clorometilcetonas de Aminoácidos/farmacologia , Caspase 1/metabolismo , Inibidores de Caspase/farmacologia , Piroptose/efeitos dos fármacos , Sepse/tratamento farmacológico , Injúria Renal Aguda/metabolismo , Animais , Antígenos CD/biossíntese , Antígenos de Diferenciação Mielomonocítica/biossíntese , Nitrogênio da Ureia Sanguínea , Creatinina , Citocinas/metabolismo , Interleucina-18/biossíntese , Interleucina-1beta/biossíntese , Interleucina-6/biossíntese , Rim/metabolismo , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/metabolismo , Sepse/metabolismoRESUMO
Aluminum-containing adjuvants have been used for nearly 100 years to enhance immune responses in billions of doses of vaccines. To date, only a few adjuvants have been approved for use in humans, among which aluminum-containing adjuvants are the only ones widely used. However, the medical need for potent and safe adjuvants is currently continuously increasing, especially those triggering cellular immune responses for cytotoxic T lymphocyte activation, which are urgently needed for the development of efficient virus and cancer vaccines. Manganese is an essential micronutrient required for diverse biological activities, but its functions in immunity remain undefined. We previously reported that Mn2+ is important in the host defense against cytosolic dsDNA by facilitating cGAS-STING activation and that Mn2+ alone directly activates cGAS independent of dsDNA, leading to an unconventional catalytic synthesis of 2'3'-cGAMP. Herein, we found that Mn2+ strongly promoted immune responses by facilitating antigen uptake, presentation, and germinal center formation via both cGAS-STING and NLRP3 activation. Accordingly, a colloidal manganese salt (Mn jelly, MnJ) was formulated to act not only as an immune potentiator but also as a delivery system to stimulate humoral and cellular immune responses, inducing antibody production and CD4+/CD8+ T-cell proliferation and activation by either intramuscular or intranasal immunization. When administered intranasally, MnJ also worked as a mucosal adjuvant, inducing high levels of secretory IgA. MnJ showed good adjuvant effects for all tested antigens, including T cell-dependent and T cell-independent antigens, such as bacterial capsular polysaccharides, thus indicating that it is a promising adjuvant candidate.
Assuntos
Adjuvantes Imunológicos/farmacologia , Manganês/farmacologia , Sais/farmacologia , Animais , Apresentação de Antígeno/efeitos dos fármacos , Antivirais/farmacologia , Vacinas Anticâncer/imunologia , Linhagem Celular , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Humanos , Interleucina-1/biossíntese , Interleucina-18/biossíntese , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Nucleotidiltransferases/metabolismo , Subunidades Proteicas/metabolismo , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologiaRESUMO
Cholesteatoma constitutes an acquired benign epidermal nonpermanent bone lesion that is locally destructive and patients often relapse. Inflammasomes, which mediate the maturation and production of IL18 and IL1ß, resulting in pyroptosis, have been documented to serve a core function in multiple inflammatory conditions. Absent in melanoma 2 (AIM2) is an inflammasome that identifies cytoplasmic DNA and has previously been reported as a pivotal modulator of inflammatory responses. Therefore, the present study aimed to determine the expression levels of AIM2 in human cholesteatoma tissues, and elucidate its function in modulating cytokine production. The expression levels of IL18, apoptosisassociated specklike protein containing a CARD (ASC), IL1ß, AIM2 and caspase1 were markedly elevated in cholesteatoma tissues. Protein expression levels of AIM2, caspase1 and ASC were localized in the cellular cytoplasm, primarily in the granular and pricklecell layers in the cholesteatoma epithelium. Induction using IFNγ, as well as cytoplasmic DNA markedly activated the AIM2 inflammasome and elevated the release of IL18 and IL1ß in human cholesteatoma keratinocytes. IFNγ was found to enhance poly(dA:dT)induced pyroptosis of cells and cytokine production. The results of the present study revealed that AIM2 expressed in human cholesteatoma serves a vital function in the inflammatory response by initiating the inflammasome signaling cascade in cholesteatoma.
Assuntos
Neoplasias Ósseas/genética , Colesteatoma/genética , Proteínas de Ligação a DNA/genética , Interleucina-18/genética , Interleucina-1beta/genética , Animais , Apoptose/efeitos dos fármacos , Neoplasias Ósseas/patologia , Proteínas Adaptadoras de Sinalização CARD/genética , Caspase 1/genética , Colesteatoma/patologia , Citocinas/biossíntese , Citocinas/genética , Citoplasma/genética , DNA/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Inflamassomos/genética , Interferon gama/genética , Interleucina-18/biossíntese , Interleucina-1beta/biossíntese , Queratinócitos/metabolismo , Neoplasias/genética , Neoplasias/patologia , Poli dA-dT/farmacologia , Piroptose/efeitos dos fármacos , Piroptose/genéticaRESUMO
Mucosal barrier immunity is essential for the maintenance of the commensal microflora and combating invasive bacterial infection. Although immune and epithelial cells are thought to be the canonical orchestrators of this complex equilibrium, here, we show that the enteric nervous system (ENS) plays an essential and non-redundant role in governing the antimicrobial protein (AMP) response. Using confocal microscopy and single-molecule fluorescence in situ mRNA hybridization (smFISH) studies, we observed that intestinal neurons produce the pleiotropic cytokine IL-18. Strikingly, deletion of IL-18 from the enteric neurons alone, but not immune or epithelial cells, rendered mice susceptible to invasive Salmonella typhimurium (S.t.) infection. Mechanistically, unbiased RNA sequencing and single-cell sequencing revealed that enteric neuronal IL-18 is specifically required for homeostatic goblet cell AMP production. Together, we show that neuron-derived IL-18 signaling controls tissue-wide intestinal immunity and has profound consequences on the mucosal barrier and invasive bacterial killing.
Assuntos
Imunidade nas Mucosas/imunologia , Interleucina-18/imunologia , Mucosa Intestinal/imunologia , Animais , Citocinas/imunologia , Sistema Nervoso Entérico/imunologia , Sistema Nervoso Entérico/metabolismo , Células Epiteliais/imunologia , Feminino , Células Caliciformes/imunologia , Interleucina-18/biossíntese , Mucosa Intestinal/metabolismo , Intestino Delgado/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/imunologia , Ratos , Ratos Sprague-Dawley , Infecções por Salmonella/imunologia , Salmonella typhimurium/imunologia , Transdução de Sinais/imunologiaRESUMO
Multiple studies have addressed the vital role of Nod-like receptor protein 3(NLRP3)/caspase-1/IL-1ß signaling in asthma. Yet, the role of NLRP3/caspase-1 in toluene diisocyanate (TDI)-induced asthma is still obscure. The aim of this study is to investigate the role of the NLRP3/caspase-1 axis in TDI-induced asthma. Using an established murine model of TDI-induced asthma as described previously, we gave the asthmatic mice a highly selective NLRP3 inhibitor, MCC950, as well as the specific caspase-1 inhibitors VX-765 and Ac-YVAD-CHO for therapeutic purposes. Airway resistance was measured and bronchoalveolar lavage fluid was analyzed. Lungs were examined by histology, immunohistochemistry, Western blotting, and flow cytometry. TDI exposure elevated the expression of NLRP3 and caspase-1 that was coupled with increased airway hyperresponsiveness (AHR), neutrophil-dominated cell infiltration, pronounced goblet cell metaplasia, extensive collagen deposition, and increased TH2/TH17 responses. Both VX-765 and Ac-YVAD-CHO effectively inhibited the activation of caspase-1 in TDI-asthmatic mice that was accompanied by dramatic attenuation of AHR, airway inflammation, and airway remodeling, in addition to a decreased TH2 response and lower levels of IL-18 and IL-1ß. MCC950 blocked the activation of NLRP3 and downregulated protein expression of caspase-1, IL-1ß, and IL-18 in TDI-exposed mice. Furthermore, MCC950 remarkably alleviated AHR, airway inflammation, airway remodeling, and significantly suppressed TH2/TH17 responses. These findings suggested that blockade of the NLRP3/caspase-1 axis effectively prevents the progression of TDI-induced asthma and could be used as therapeutic targets for asthmatics.
Assuntos
Asma/tratamento farmacológico , Compostos Heterocíclicos de 4 ou mais Anéis/uso terapêutico , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidores , Serpinas/uso terapêutico , Sulfonas/uso terapêutico , Tolueno 2,4-Di-Isocianato/toxicidade , Proteínas Virais/uso terapêutico , Remodelação das Vias Aéreas/efeitos dos fármacos , Animais , Asma/induzido quimicamente , Asma/imunologia , Caspase 1/fisiologia , Modelos Animais de Doenças , Furanos , Indenos , Interleucina-18/biossíntese , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/fisiologia , Hipersensibilidade Respiratória/tratamento farmacológico , Sulfonamidas , Células Th17/imunologia , Células Th2/imunologiaRESUMO
INTRODUCTION: Perinatal hypoxic-ischemic (HI) insult is an important cause of brain injury in neonates. The development of novel treatment strategies for neonates with HI brain injury is urgently needed. Ginkgolide B (GB) is a main component of Ginkgo biloba extracts with a long history of use in traditional Chinese medicine. However, it is unknown whether GB could play a protective role in hypoxic stress in immature animals. METHODS: Using neonatal hypoxic-ischemic (HI) brain injury model of rat pups, neurological score, infarct size, and brain edema were evaluated after HI injury. The activation of microglia and the production of IL-1ß and IL-18 were detected by immunohistochemistry and ELISA, respectively. A priming signal (NF-κB P65) and an activation signal (Caspase-1) of NLRP3 inflammasome activation were detected by western blot analyses. RESULTS: GB administrated 30 min prior to ischemia induction can improve neurological disorder, reduce infarct volume and alleviate cerebral edema. Compared with the HI groups, GB inhibited the activation of microglia and decreased the production of IL-1ß and IL-18 in neocortex. Furthermore, GB reduced NLRP3 expression mainly in microglia, and significantly inhibited the expression of Caspase-1 and the nuclear translocation of NF-κB P65, preventing NLRP3 inflammasome activation. CONCLUSIONS: GB ameliorates hypoxic-ischemic brain injury in the neonatal male rat via inhibiting NLRP3 inflammasome activation.
Assuntos
Sequestradores de Radicais Livres/farmacologia , Ginkgolídeos/farmacologia , Hipóxia-Isquemia Encefálica/psicologia , Inflamassomos/efeitos dos fármacos , Lactonas/farmacologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Comportamento Animal , Edema Encefálico/etiologia , Edema Encefálico/patologia , Infarto Encefálico/patologia , Caspase 1/biossíntese , Caspase 1/efeitos dos fármacos , Feminino , Hipóxia-Isquemia Encefálica/patologia , Interleucina-18/biossíntese , Interleucina-1beta/biossíntese , Ativação de Macrófagos/efeitos dos fármacos , Masculino , Microglia/efeitos dos fármacos , Gravidez , Ratos , Fator de Transcrição RelA/biossínteseRESUMO
Quercetin, a polyphenol, belongs to a class of flavonoids that exerts anti-inflammatory effects. Interleukin (IL)-18 is a member of the IL-1 family cytokine that regulates immune responses and is implicated in various inflammatory skin diseases. Absent in melanoma 2 (AIM2) is a cytosolic double-stranded (ds) DNA sensor that recognizes the dsDNA of a microbial or host origin. Binding of dsDNA to AIM2 simulates caspase-1-dependent inflammasome activity, which leads to the production of IL-1ß and IL-18. Increased levels of AIM2 have been observed in patients with inflammatory skin diseases. In the current study, we investigated the issue of whether or how Quercetin attenuates poly (dA:dT), a synthetic analog of microbial dsDNA, -induced IL-18 secretion in IFN-γ-primed human keratinocytes. Treatment with 5 and 10⯵M of Quercetin inhibited the poly (dA:dT)-induced secretion of IL-18 after IFN-γ priming and before poly (dA:dT)-induced AIM2 activation. In addition, treatment with Quercetin at 10⯵M, significantly inhibited the phosphorylation of JAK2 and STAT1, and the nuclear translocation of phosphorylated STAT1 in poly (dA:dT)-treated and IFN-γ-primed keratinocytes. These results suggest that treatment with Quercetin inhibits the poly (dA:dT)-induced secretion of IL-18 via down-regulation of the expressions of AIM2 and pro-caspase-1 by inhibiting the JAK2/STAT1 pathway in IFN-γ-primed keratinocytes.
Assuntos
Caspase 1/metabolismo , Proteínas de Ligação a DNA/metabolismo , Interleucina-18/biossíntese , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Quercetina/farmacologia , Anti-Inflamatórios não Esteroides/farmacologia , Caspase 1/genética , Linhagem Celular , Células Cultivadas , Proteínas de Ligação a DNA/genética , Regulação para Baixo/efeitos dos fármacos , Humanos , Interferon gama/metabolismo , Interferon gama/farmacologia , Janus Quinase 2/metabolismo , Queratinócitos/imunologia , Poli dA-dT/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Interleukin-18 (IL-18) is a pleiotropic cytokine of the IL-1 family with multiple context dependent functions. We and others have shown that HIV infection is accompanied by increased circulating levels of IL-18 along with decreased levels of its antagonist, Interleukin-18 Binding Protein (IL-18BP). The infection is also accompanied by intestinal inflammation and decreased intestinal integrity as measured by intestinal permeability, regeneration and repair. However, little is known concerning the relation between high level of IL-18 associated with the viral infection and intestinal permeability. Here we demonstrate that HIV treatment increases production of IL-18 and decreases that of IL-18BP production in human intestinal epithelial cell (IEC) lines. IL-18 causes apoptosis of the IEC by activating caspase-1 and caspase-3. It induces epithelial barrier hyperpermeability by decreasing and disrupting both tight and adherens junction proteins, occludin, claudin 2 and beta-catenin. Disorganization of F-actin was also observed in the IEC that were exposed to the cytokine. Moreover IL-18 decreases transepithelial electrical resistance (TEER) in Caco-2 and increases permeability in HT29 monolayers. The cells' treatment with IL-18 causes an increase in the expression of phosphorylated myosin II regulatory light-chain (p-MLC) and myosin light-chain kinase (MLCK), and a decrease in phosphorylated Signal Transducer and Activator of Transcription (p-STAT)-5. This increase in p-MLC is suppressed by a Rho-kinase (ROCK)-specific inhibitor. Interestingly, the levels of the cytokine correlate with those of LPS in the circulation in three different categories of HIV infected patients (HAART-naïve and HAART-treated HIV-infected individuals, and Elite controls) as well as in healthy controls. Collectively, these results suggest that the HIV-induced IL-18 plays a role in increased intestinal permeability and microbial translocation observed in HIV-infected individuals.
Assuntos
Translocação Bacteriana , Células Epiteliais/metabolismo , Infecções por HIV/metabolismo , HIV-1/metabolismo , Interleucina-18/biossíntese , Mucosa Intestinal/metabolismo , Células CACO-2 , Células Epiteliais/microbiologia , Células Epiteliais/patologia , Células Epiteliais/virologia , Feminino , Infecções por HIV/microbiologia , Infecções por HIV/patologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Mucosa Intestinal/virologia , Masculino , PermeabilidadeRESUMO
The aim of this study was to characterize an in vitro modulating effect of three commensal Lactobacillus strains on cellular differentiation of non-transformed crypt-like rat small intestinal cell line IEC-18. IEC-18 was grown on extracellular matrix, with or without presence of Lactobacillus strains. Gene expression of IEC-18 bacterial detection system - such as Toll-like receptors TLR-2, TLR-4, signal adapter MyD88, cytoplasmic NOD2 receptor, inflammatory cytokines IL-18, IL-1beta, chemokine IL-8 and enzyme caspase-1 - was evaluated using real-time PCR. Expression and localization of TLR-2, TLR-4, IL-18 and caspase-1 proteins was demonstrated by Western blotting and immunofluorescent staining. Secretion of IL-18 to apical and basolateral surfaces was assayed by ELISA. Our results suggested that L. casei LOCK0919 accelerated differentiation of IEC-18 by stimulating TLR-2, TLR-4, MyD88, IL-18, caspase-1 mRNAs and proteins. L. casei LOCK0919 increased expression and transfer of villin and beta-catenin from cytoplasm to cell membrane. Presence of L. rhamnosus LOCK0900 resulted in detachment of IEC-18 layer from extracellular matrix leading to induction of IL-1beta, of TLR-2 and IL-8 mRNAs and stimulation of MyD88, caspase-1 and cytosolic receptor NOD2 mRNAs. L. rhamnosus LOCK0908 was not recognized by TLR-2 or TLR-4 receptors. Lactobacilli-IEC-18 crosstalk enhanced immune and barrier mucosal functions.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Mucosa Intestinal/citologia , Lacticaseibacillus casei , Lacticaseibacillus rhamnosus , Probióticos/farmacologia , Animais , Caspase 1/biossíntese , Citocinas/biossíntese , Regulação da Expressão Gênica/efeitos dos fármacos , Interleucina-18/biossíntese , Mucosa Intestinal/efeitos dos fármacos , Proteínas dos Microfilamentos/biossíntese , RNA Mensageiro/biossíntese , Ratos , Frações Subcelulares/metabolismo , Receptores Toll-Like/biossíntese , Receptores Toll-Like/efeitos dos fármacos , beta Catenina/biossínteseRESUMO
Interferon-γ (IFN-γ), a cytokine produced by activated natural killer cells and T lymphocytes, is an important regulator of innate and adaptive immunity. Interleukin (IL)-18, also known as IFN-γ-inducing factor, is a cytokine that induces T and natural killer cells to produce IFN-γ. In this study, the chicken IL-18 (ChIL-18) and chicken IFN-γ (ChIFN-γ) genes were inserted into the pET28a prokaryotic expression vector, resulting in pET28a-IL-18 and pET28a-IFN-γ, respectively. These plasmids were transformed into Escherichia coli strain BL21, and the ChIL-18 and ChIFN-γ proteins were expressed and purified. To determine their antiviral activities, 200 ng/mL of ChIL-18 and/or ChIFN-γ were inoculated into chicken embryonic fibroblast cells. After 24 h, one 50% tissue culture infective dose (TCID50) of infectious bursal disease virus (IBDV) was inoculated into the chicken embryonic fibroblast cells. The results showed that the antiviral effect of ChIL-18 and ChIFN-γ in combination was better than that of ChIL-18 or ChIFN-γ alone. Next, 14-day-old chicken were injected with 200 µg of ChIL-18 and/or ChIFN-γ and then were challenged with 103 TCID50 of IBDV via intraperitoneal injection. The results showed that the proliferation of IBDV was inhibited by the injection of the recombinant proteins, especially the combination of ChIL-18 and ChIFN-γ, as evidenced by cytokine detection, quantitative PCR, and pathology analyses. These results indicate that ChIL-18 and ChIFN-γ could inhibit IBDV infection and the combination of ChIL-18 and ChIFN-γ has a better inhibitory effect than either cytokine alone.
Assuntos
Infecções por Birnaviridae/prevenção & controle , Vírus da Doença Infecciosa da Bursa/imunologia , Interferon gama/genética , Interleucina-18/genética , Replicação Viral/imunologia , Animais , Antivirais/metabolismo , Infecções por Birnaviridae/imunologia , Infecções por Birnaviridae/virologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Células Cultivadas , Embrião de Galinha , Galinhas , Escherichia coli/genética , Escherichia coli/metabolismo , Interferon gama/biossíntese , Interferon gama/imunologia , Interleucina-18/biossíntese , Interleucina-18/imunologia , Células Matadoras Naturais/imunologia , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Plasmídeos/genética , Replicação Viral/genéticaRESUMO
The Gram-negative bacterial cell wall component lipopolysaccharide (LPS) is recognized by the noncanonical inflammasome protein caspase-11 in the cytosol of infected host cells and thereby prompts an inflammatory immune response linked to sepsis. Host guanylate binding proteins (GBPs) promote infection-induced caspase-11 activation in tissue culture models, and yet their in vivo role in LPS-mediated sepsis has remained unexplored. LPS can be released from lysed bacteria as "free" LPS aggregates or actively secreted by live bacteria as a component of outer membrane vesicles (OMVs). Here, we report that GBPs control inflammation and sepsis in mice injected with either free LPS or purified OMVs derived from Gram-negative Escherichia coli In agreement with our observations from in vivo experiments, we demonstrate that macrophages lacking GBP2 expression fail to induce pyroptotic cell death and proinflammatory interleukin-1ß (IL-1ß) and IL-18 secretion when exposed to OMVs. We propose that in order to activate caspase-11 in vivo, GBPs control the processing of bacterium-derived OMVs by macrophages as well as the processing of circulating free LPS by as-yet-undetermined cell types.IMPORTANCE The bacterial cell wall component LPS is a strong inducer of inflammation and is responsible for much of the toxicity of Gram-negative bacteria. Bacteria shed some of their cell wall and its associated LPS in the form of outer membrane vesicles (OMVs). Recent work demonstrated that secreted OMVs deliver LPS into the host cell cytosol by an unknown mechanism, resulting in the activation of the proinflammatory LPS sensor caspase-11. Here, we show that activation of cytosolic caspase-11 by OMVs requires additional host factors, the so-called guanylate binding proteins (GBPs). The discovery of GBPs as regulators of OMV-mediated inflammation paves the way toward a mechanistic understanding of the host response toward bacterial OMVs and may lead to effective strategies to ameliorate inflammation induced by bacterial infections.
Assuntos
Proteínas da Membrana Bacteriana Externa/imunologia , Escherichia coli/patogenicidade , Proteínas de Ligação ao GTP/metabolismo , Inflamassomos/imunologia , Inflamassomos/metabolismo , Lipopolissacarídeos/metabolismo , Macrófagos/metabolismo , Macrófagos/microbiologia , Animais , Proteínas da Membrana Bacteriana Externa/administração & dosagem , Proteínas da Membrana Bacteriana Externa/genética , Caspases/metabolismo , Caspases Iniciadoras , Células Cultivadas , Citosol/metabolismo , Ativação Enzimática , Inflamação , Interleucina-18/biossíntese , Interleucina-1beta/biossíntese , Lipopolissacarídeos/imunologia , Camundongos , Piroptose , Vesículas Secretórias/metabolismoRESUMO
Leptin is a cytokinelike hormone secreted by adipocytes, which serves to control energy expenditure and metabolism. In addition, leptin may modulate the innate and adaptive immune responses. The innate immune cell sensor nucleotidebinding oligomerization domainlike receptor family pyrin domaincontaining 3 (NLRP3) inflammasome is mainly expressed in myeloid immune cells, including macrophages. The NLRP3 inflammasome serves a pivotal role in the development and maintenance of autoimmunity and inflammation. The expression levels of caspase1, apoptosisassociated specklike protein containing a CARD, interleukin (IL)18, IL1ß and leptin are significantly reduced in the white adipose tissue of nonsteroidal antiinflammatory drugactivated gene1 transgenic mice. However, the association between leptin and the NLRP3 inflammasome has not yet to be determined. The aim of the present study, was to explore the role of leptin on NLRP3 inflammasome. In order to do this, IL1ß and IL18 expression levels were investigated in RAW 264.7 cells after incubation with leptin of increasing doses by Elisa or reverse transcriptionquantitative polymerase chain reaction, and to assess whether IL1ß and IL18 were affected after caspase1 activity being inhibited by an inhibitor or by silencing NLRP3 expression. The results of the present study demonstrated that leptin enhanced the mRNA and protein expression levels of IL18 in RAW 264.7 cells via activation of the NLRP3 inflammasome. This is achieved partly by enhancing the production of reactive oxygen species and K+ efflux. Therefore, leptin may be considered a novel activator and modulator of the NLRP3 inflammasome.
Assuntos
Inflamassomos/metabolismo , Interleucina-18/biossíntese , Leptina/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Animais , Biomarcadores , Citocinas/metabolismo , Técnicas de Inativação de Genes , Mediadores da Inflamação/metabolismo , Macrófagos/metabolismo , Camundongos , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Células RAW 264.7 , Espécies Reativas de Oxigênio/metabolismoRESUMO
BACKGROUND: Acute pancreatitis is an inflammatory disorder of the pancreas that is responsible for significant morbidity and mortality. The inflammasome pathway has acquired significant relevance in the pathogenesis of many inflammatory disorders, but its role in patients with acute pancreatitis still awaits clarification. METHODS: We performed a prospective study in which 27 patients with acute pancreatitis and 16 healthy controls were included. We isolated peripheral blood mononuclear cells (PBMCs) and we assessed the expression and activation of different inflammasomes as well as their association with the clinical course of the disease. RESULTS: Our results show that PBMCs from patients with acute pancreatitis have elevated expression of several components of the inflammasome complex, including the inflammasome-forming receptor absent in melanoma 2 (AIM2), early during the onset of the disease. Activation of the AIM2 or NLRP3 inflammasomes in PBMCs from patients with acute pancreatitis results in exacerbated IL-1ß and IL-18 production compared with PBMCs from healthy controls. Furthermore, both AIM2 mRNA expression and AIM2-mediated production of IL-1ß by PBMCs correlated with increased systemic inflammation in these patients. Last, AIM2 expression was further increased in those patients that developed transient or persistent organ failure (moderate or severe acute pancreatitis). CONCLUSIONS: Our data demonstrates that AIM2 inflammasome expression and activation is increased early during the course of acute pancreatitis, and suggests that AIM2 activation may affect systemic inflammation and organ failure in these patients.
Assuntos
Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/metabolismo , Inflamassomos , Pancreatite/patologia , Doença Aguda , Adulto , Idoso , Feminino , Humanos , Imunidade Celular , Inflamação/patologia , Interleucina-18/biossíntese , Interleucina-18/genética , Interleucina-1beta/biossíntese , Interleucina-1beta/genética , Masculino , Pessoa de Meia-Idade , Monócitos , Estudos Prospectivos , RNA Mensageiro/biossíntese , RNA Mensageiro/genéticaRESUMO
BACKGROUND: Canakinumab is a human anti-interleukin-1ß (IL-1ß) monoclonal antibody neutralizing IL-1ß-mediated pathways. We sought to characterize the molecular response to canakinumab and evaluate potential markers of response using samples from two pivotal trials in systemic juvenile idiopathic arthritis (SJIA). METHODS: Gene expression was measured in patients with febrile SJIA and in matched healthy controls by Affymetrix DNA microarrays. Transcriptional response was assessed by gene expression changes from baseline to day 3 using adapted JIA American College of Rheumatology (aACR) response criteria (50 aACR JIA). Changes in pro-inflammatory cytokines IL-6 and IL-18 were assessed up to day 197. RESULTS: Microarray analysis identified 984 probe sets differentially expressed (≥2-fold difference; P < 0.05) in patients versus controls. Over 50% of patients with ≥50 aACR JIA were recognizable by baseline expression values. Analysis of gene expression profiles from patients achieving ≥50 aACR JIA response at day 15 identified 102 probe sets differentially expressed upon treatment (≥2-fold difference; P < 0.05) on day 3 versus baseline, including IL-1ß, IL-1 receptors (IL1-R1 and IL1-R2), IL-1 receptor accessory protein (IL1-RAP), and IL-6. The strongest clinical response was observed in patients with higher baseline expression of dysregulated genes and a strong transcriptional response on day 3. IL-6 declined by day 3 (≥8-fold decline; P < 0.0001) and remained suppressed. IL-18 declined on day 57 (≥1.5-fold decline, P ≤ 0.002). CONCLUSIONS: Treatment with canakinumab in SJIA patients resulted in downregulation of innate immune response genes and reductions in IL-6 and clinical symptoms. Additional research is needed to investigate potential differences in the disease mechanisms in patients with heterogeneous gene transcription profiles. TRIAL REGISTRATION: Clinicaltrials.gov: NCT00886769 (trial 1). Registered on 22 April 2009; NCT00889863 (trial 2). Registered on 21 April 2009.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Artrite Juvenil/tratamento farmacológico , Interleucina-18/biossíntese , Interleucina-6/biossíntese , Transcriptoma/efeitos dos fármacos , Adolescente , Anticorpos Monoclonais Humanizados , Criança , Pré-Escolar , Regulação para Baixo , Feminino , Perfilação da Expressão Gênica , Humanos , Imunoensaio , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Adulto JovemRESUMO
OBJECTIVE: Western lifestyle and diet are major environmental factors playing a role in the development of IBD. Titanium dioxide (TiO2) nanoparticles are widely used as food additives or in pharmaceutical formulations and are consumed by millions of people on a daily basis. We investigated the effects of TiO2 in the development of colitis and the role of the nucleotide-binding oligomerisation domain receptor, pyrin domain containing (NLRP)3 inflammasome. DESIGN: Wild-type and NLRP3-deficient mice with dextran sodium sulfate-induced colitis were orally administered with TiO2 nanoparticles. The proinflammatory effects of TiO2 particles in cultured human intestinal epithelial cells (IECs) and macrophages were also studied, as well as the ability of TiO2 crystals to traverse IEC monolayers and accumulate in the blood of patients with IBD using inductively coupled plasma mass spectrometry. RESULTS: Oral administration of TiO2 nanoparticles worsened acute colitis through a mechanism involving the NLRP3 inflammasome. Importantly, crystals were found to accumulate in spleen of TiO2-administered mice. In vitro, TiO2 particles were taken up by IECs and macrophages and triggered NLRP3-ASC-caspase-1 assembly, caspase-1 cleavage and the release of NLRP3-associated interleukin (IL)-1ß and IL-18. TiO2 also induced reactive oxygen species generation and increased epithelial permeability in IEC monolayers. Increased levels of titanium were found in blood of patients with UC having active disease. CONCLUSION: These findings indicate that individuals with a defective intestinal barrier function and pre-existing inflammatory condition, such as IBD, might be negatively impacted by the use of TiO2 nanoparticles.
Assuntos
Colite/imunologia , Corantes/efeitos adversos , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR , Nanopartículas/efeitos adversos , Titânio/efeitos adversos , Animais , Caspase 1/metabolismo , Colite/induzido quimicamente , Colite/metabolismo , Corantes/administração & dosagem , Sulfato de Dextrana/efeitos adversos , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Humanos , Interleucina-18/biossíntese , Interleucina-1beta/metabolismo , Intestinos/citologia , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nanopartículas/administração & dosagem , Espécies Reativas de Oxigênio/metabolismo , Baço/patologia , Titânio/administração & dosagem , Titânio/sangueRESUMO
Individuals suffering from autoimmune disorders possess a hyperactive cellular phenotype where tolerance to self-antigens is lost. Autophagy has been implicated in both the induction and prevention of autoimmunity, and modulators of this cellular recycling process hold high potential for the treatment of autoimmune diseases. In this study, we determine the effects of a loss of autophagy in dendritic cells (DCs), as well as both B cells and DCs, in a TLR7-mediated model of autoimmunity, similar to systemic lupus erythematosus, where both cell types are critical for disease. Although a loss of DC autophagy slowed disease, the combined loss of autophagy in both cell types resulted in a lethal sepsis-like environment, which included tissue inflammation and hyperproduction of inflammasome-associated cytokines. Ablation of B cell signaling reversed this phenotype, indicating that activation of these cells is an essential step in disease induction. Thus, autophagy plays a dichotomous role in this model of disease.
Assuntos
Autoimunidade , Autofagia/fisiologia , Linfócitos B/fisiologia , Células Dendríticas/fisiologia , Inflamação/etiologia , Glicoproteínas de Membrana/fisiologia , Receptor 7 Toll-Like/fisiologia , Animais , Interleucina-18/biossíntese , Lúpus Eritematoso Sistêmico/etiologia , Linfadenopatia/etiologia , Camundongos , Camundongos Endogâmicos C57BL , Esplenomegalia/etiologiaRESUMO
Vitamin B6 includes six water-soluble vitamers: pyridoxal (PL), pyridoxamine (PM), pyridoxine (PN), and their phosphorylated forms. Pyridoxal 5'-phosphate (PLP) is an important cofactor for many metabolic enzymes. Several lines of evidence demonstrate that blood levels of PLP are significantly lower in patients with inflammation than in control subjects and that vitamin B6 has anti-inflammatory effects, with therapeutic potential for a variety of inflammatory diseases. Although one of our group previously demonstrated that PL inhibits the NF-κB pathway, the molecular mechanism by which vitamin B6 suppresses inflammation is not well understood. Here, we showed that both PL and PLP suppressed the expression of cytokine genes in macrophages by inhibiting Toll-like receptor (TLR)-mediated TAK1 phosphorylation and the subsequent NF-κB and JNK activation. Furthermore, PL and PLP abolished NLRP3-dependent caspase-1 processing and the subsequent secretion of mature IL-1ß and IL-18 in LPS-primed macrophages. In contrast, PM and PN had little effect on IL-1ß production. PLP, but not PL, markedly reduced the production of mitochondrial reactive oxygen species (ROS) in peritoneal macrophages. Importantly, PL and PLP reduced IL-1ß production induced by LPS and ATP, or by LPS alone, in mice. Moreover, PL and PLP protected mice from lethal endotoxic shock. Collectively, these findings reveal novel anti-inflammatory activities for vitamin B6 and suggest its potential for preventing inflammatory diseases driven by the NLRP3 inflammasome.
Assuntos
Anti-Inflamatórios/farmacologia , Inflamassomos/metabolismo , Interleucina-1beta/biossíntese , Macrófagos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Vitamina B 6/farmacologia , Animais , Interleucina-18/biossíntese , Lipopolissacarídeos/toxicidade , MAP Quinase Quinase Quinases/metabolismo , Camundongos , Camundongos Endogâmicos ICR , NF-kappa B/metabolismo , Fosforilação/efeitos dos fármacos , Choque Séptico/induzido quimicamente , Choque Séptico/metabolismo , Choque Séptico/prevenção & controleRESUMO
Severe hepatic inflammation is a common cause of acute or chronic liver disease. Macrophages are one of the key mediators which regulate the progress of hepatic inflammation. Increasing evidence shows that the TAM (TYRO3, AXL and MERTK) family of RTKs (receptor tyrosine kinases), which is expressed in macrophages, alleviates inflammatory responses through a negative feedback loop. However, the functional contribution of each TAM family member to the progression of hepatic inflammation remains elusive. In this study, we explore the role of individual TAM family proteins during autophagy induction and evaluate their contribution to hepatic inflammation. Among the TAM family of RTKs, AXL (AXL receptor tyrosine kinase) only induces autophagy in macrophages after interaction with its ligand, GAS6 (growth arrest specific 6). Based on our results, autophosphorylation of 2 tyrosine residues (Tyr815 and Tyr860) in the cytoplasmic domain of AXL in mice is required for autophagy induction and AXL-mediated autophagy induction is dependent on MAPK (mitogen-activated protein kinase)14 activity. Furthermore, induction of AXL-mediated autophagy prevents CASP1 (caspase 1)-dependent IL1B (interleukin 1, ß) and IL18 (interleukin 18) maturation by inhibiting NLRP3 (NLR family, pyrin domain containing 3) inflammasome activation. In agreement with these observations, axl-/- mice show more severe symptoms than do wild-type (Axl+/+) mice following acute hepatic injury induced by administration of lipopolysaccharide (LPS) or carbon tetrachloride (CCl4). Hence, GAS6-AXL signaling-mediated autophagy induction in murine macrophages ameliorates hepatic inflammatory responses by inhibiting NLRP3 inflammasome activation.