RESUMEN
Pulmonary emphysema is associated with dysregulated innate immune responses that promote chronic pulmonary inflammation and alveolar apoptosis, culminating in lung destruction. However, the molecular regulators of innate immunity that promote emphysema are ill-defined. Here, we investigated whether innate immune inflammasome complexes, comprising the adaptor ASC, Caspase-1 and specific pattern recognition receptors (PRRs), promote the pathogenesis of emphysema. In the lungs of emphysematous patients, as well as spontaneous gp130F/F and cigarette smoke (CS)-induced mouse models of emphysema, the expression (messenger RNA and protein) and activation of ASC, Caspase-1, and the inflammasome-associated PRR and DNA sensor AIM2 were up-regulated. AIM2 up-regulation in emphysema coincided with the biased production of the mature downstream inflammasome effector cytokine IL-1ß but not IL-18. These observations were supported by the genetic blockade of ASC, AIM2, and the IL-1 receptor and therapy with AIM2 antagonistic suppressor oligonucleotides, which ameliorated emphysema in gp130F/F mice by preventing elevated alveolar cell apoptosis. The functional requirement for AIM2 in driving apoptosis in the lung epithelium was independent of its expression in hematopoietic-derived immune cells and the recruitment of infiltrating immune cells in the lung. Genetic and inhibitor-based blockade of AIM2 also protected CS-exposed mice from pulmonary alveolar cell apoptosis. Intriguingly, IL-6 trans-signaling via the soluble IL-6 receptor, facilitated by elevated levels of IL-6, acted upstream of the AIM2 inflammasome to augment AIM2 expression in emphysema. Collectively, we reveal cross-talk between the AIM2 inflammasome/IL-1ß and IL-6 trans-signaling axes for potential exploitation as a therapeutic strategy for emphysema.
Asunto(s)
Proteínas de Unión al ADN , Inmunidad Innata , Interleucina-1beta , Interleucina-6 , Enfisema Pulmonar , Animales , Apoptosis , Caspasa 1/metabolismo , Receptor gp130 de Citocinas/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Humanos , Inflamasomas/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Ratones , Enfisema Pulmonar/inmunologíaRESUMEN
OBJECTIVE: The absent in melanoma 2 (AIM2) cytosolic pattern recognition receptor and DNA sensor promotes the pathogenesis of autoimmune and chronic inflammatory diseases via caspase-1-containing inflammasome complexes. However, the role of AIM2 in cancer is ill-defined. DESIGN: The expression of AIM2 and its clinical significance was assessed in human gastric cancer (GC) patient cohorts. Genetic or therapeutic manipulation of AIM2 expression and activity was performed in the genetically engineered gp130 F/F spontaneous GC mouse model, as well as human GC cell line xenografts. The biological role and mechanism of action of AIM2 in gastric tumourigenesis, including its involvement in inflammasome activity and functional interaction with microtubule-associated end-binding protein 1 (EB1), was determined in vitro and in vivo. RESULTS: AIM2 expression is upregulated by interleukin-11 cytokine-mediated activation of the oncogenic latent transcription factor STAT3 in the tumour epithelium of GC mouse models and patients with GC. Genetic and therapeutic targeting of AIM2 in gp130 F/F mice suppressed tumourigenesis. Conversely, AIM2 overexpression augmented the tumour load of human GC cell line xenografts. The protumourigenic function of AIM2 was independent of inflammasome activity and inflammation. Rather, in vivo and in vitro AIM2 physically interacted with EB1 to promote epithelial cell migration and tumourigenesis. Furthermore, upregulated expression of AIM2 and EB1 in the tumour epithelium of patients with GC was independently associated with poor patient survival. CONCLUSION: AIM2 can play a driver role in epithelial carcinogenesis by linking cytokine-STAT3 signalling, innate immunity and epithelial cell migration, independent of inflammasome activation.
Asunto(s)
Melanoma , Neoplasias Gástricas , Animales , Carcinogénesis/genética , Movimiento Celular/genética , Receptor gp130 de Citocinas/metabolismo , ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Humanos , Inmunidad Innata/genética , Inflamasomas/genética , Inflamasomas/metabolismo , Ratones , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Neoplasias Gástricas/patología , Regulación hacia ArribaRESUMEN
Cytosolic DNA receptor cyclic GMP-AMP (cGAMP) synthase (cGAS) has been shown to be critically involved in the detection of cytosolic, self- and non-self-DNA, initiating a type I IFN response through the adaptor protein Stimulator of Interferon Genes (STING) and interferon regulatory factor 3 (IRF3). Current studies propose that canonical binding of dsDNA by cGAS depends on DNA length, but not on base sequence. In contrast, activation of TLR9 is sequence dependent. It requires unmethylated CpG dinucleotides in microbial DNA, which is mimicked by synthetic oligodeoxynucleotides (ODN). Here, we provide evidence that d-type ODN (D-ODN), but not K-type ODN (K-ODN), bind to human cGAS and activate downstream signaling. Transfection of D-ODN into a TLR9-deficient, human monocytic cell line (THP-1) induced phosphorylation of IRF3 and secretion of IFN. This response was absent in cells with CRISPR/Cas9-mediated cGAS- or STING-deficiency. Utilizing a protein pulldown approach, we further demonstrate direct binding of D-ODN to cGAS. Induction of a type I IFN response by D-ODN was confirmed in human primary monocytes and monocyte-derived macrophages. These results are relevant to our understanding of self-nonself-discrimination by cGAS and to the pharmacologic effects of ODN, which currently are investigated in clinical studies.
Asunto(s)
Citosol/inmunología , Interferón Tipo I/inmunología , Proteínas de la Membrana/inmunología , Nucleótidos Cíclicos/inmunología , Oligodesoxirribonucleótidos/inmunología , Transducción de Señal/inmunología , Células Cultivadas , Células HEK293 , Humanos , Factor 3 Regulador del Interferón/inmunología , Macrófagos/inmunología , Monocitos/inmunología , Fosforilación/inmunología , Células THP-1RESUMEN
Immune-mediated inflammation must be down-regulated to facilitate tissue remodeling during homeostatic restoration of an inflammatory response. Uncontrolled or over-exuberant immune activation can cause autoimmune diseases, as well as tissue destruction. A151, the archetypal example of a chemically synthesized suppressive oligodeoxynucleotide (ODN) based on repetitive telomere-derived TTAGGG sequences, was shown to successfully down-regulate a variety of immune responses. However, the degree, duration and breadth of A151-induced transcriptome alterations remain elusive. Here, we performed a comprehensive microarray analysis in combination with Ingenuity Pathway Analysis (IPA) using murine splenocytes to investigate the underlying mechanism of A151-dependent immune suppression. Our results revealed that A151 significantly down-regulates critical mammalian target of rapamycin (mTOR) activators (Pi3kcd, Pdpk1 and Rheb), elements downstream of mTOR signaling (Rps6ka1, Myc, Stat3 and Slc2a1), an important component of the mTORC2 protein complex (Rictor) and Mtor itself. The effects of A151 on mTOR signaling were dose- and time-dependent. Moreover, flow cytometry and immunoblotting analyses demonstrated that A151 is able to reverse mTOR phosphorylation comparably to the well-known mTOR inhibitor rapamycin. Furthermore, Seahorse metabolic assays showed an A151 ODN-induced decrease in both oxygen consumption and glycolysis implying that a metabolically inert state in macrophages could be triggered by A151 treatment. Overall, our findings suggested novel insights into the mechanism by which the immune system is metabolically modulated by A151 ODN.
Asunto(s)
Inmunosupresores/farmacología , Oligodesoxirribonucleótidos/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Secuencias de Aminoácidos/efectos de los fármacos , Animales , Células Cultivadas , Ratones , Ratones Endogámicos C57BL , Oligodesoxirribonucleótidos/farmacología , Fosforilación/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Toll-like receptors (TLR) trigger the immune system to mount a rapid innate response capable of protecting the host from a wide variety of bacterial and viral pathogens. There is interest in harnessing TLR agonists to reduce the susceptibility of at-risk populations to infection. However, the widespread prophylactic use of TLR agonists has been compromised by the need to administer them by parenteral injection. An exception is the TLR7/8 agonist R848, which can boost gastrointestinal and systemic immunity when administered orally. This work examines the effect of R848 on host susceptibility to Listeria monocytogenes in a murine challenge model and describes the underlying mechanisms. Results show that prophylactic administration of R848 significantly reduces susceptibility to infection of BALB/c mice, an effect that lasts 1 week. Oral R848 directly stimulated B cells to produce cytokines and Ig. In the absence of B cells, R848-mediated protection was lost. These findings support the use of oral R848 to reduce the susceptibility of at-risk individuals to infection and identify the critical role of B cells in TLR7-mediated resistance to bacterial infection.
Asunto(s)
Linfocitos B/inmunología , Imidazoles/farmacología , Listeria monocytogenes/inmunología , Listeriosis/prevención & control , Glicoproteínas de Membrana/inmunología , Receptor Toll-Like 7/inmunología , Animales , Listeriosis/inmunología , Ratones , Ratones Endogámicos BALB CRESUMEN
Systematic Lupus Erythematosus (SLE) is an autoimmune syndrome of unclear etiology. While T and B cell abnormalities contribute to disease pathogenesis, recent work suggests that inflammatory M1-like macrophages also play a role. Previous work showed that the TLR2/1 agonist PAM3CSK4 (PAM3) could stimulate normal human monocytes to preferentially differentiate into immunosuppressive M2-like rather than inflammatory M1-like macrophages. This raised the possibility of PAM3 being used to normalize the M1:M2 ratio in SLE. Consistent with that possibility, monocytes from lupus patients differentiated into M2-like macrophages when treated with PAM3 in vitro. Furthermore, lupus-prone NZB x NZW F1 mice responded similarly to weekly PAM3 treatment. Normalization of the M2 macrophage frequency was associated with delayed disease progression, decreased autoantibody and inflammatory cytokine synthesis, reduced proteinuria and prolonged survival in NZB x NZW F1 mice. The ability of PAM3 to bias monocyte differentiation in favor of immunosuppressive macrophages may represent a novel approach to the therapy of SLE.
Asunto(s)
Lipopéptidos/farmacología , Lupus Eritematoso Sistémico/etiología , Lupus Eritematoso Sistémico/metabolismo , Activación de Macrófagos/efectos de los fármacos , Activación de Macrófagos/inmunología , Macrófagos/inmunología , Monocitos/inmunología , Animales , Plasticidad de la Célula/efectos de los fármacos , Plasticidad de la Célula/inmunología , Citocinas/metabolismo , Endocitosis/inmunología , Femenino , Inmunofenotipificación , Lupus Eritematoso Sistémico/complicaciones , Nefritis Lúpica/etiología , Nefritis Lúpica/metabolismo , Nefritis Lúpica/patología , Macrófagos/metabolismo , Masculino , Ratones , Monocitos/metabolismo , Receptor Toll-Like 1/metabolismo , Receptor Toll-Like 2/metabolismoRESUMEN
Th cells sensitized against autoantigens acquire pathogenicity following two sequential events, namely activation by their target Ag and a process named "licensing." In this study, we analyzed these processes in a transgenic mouse system in which TCR-transgenic Th cells specific to hen egg lysozyme (HEL) are adoptively transferred to recipients and induce inflammation in eyes expressing HEL. Our data show that the notion that the lung is the organ where "licensing" for pathogenicity takes place is based on biased data collected with cells injected i.v., a route in which most transferred cells enter via the lung. Thus, we found that when donor cells were activated in vitro and injected intraperitoneally, or were activated in vivo, they migrated simultaneously to the lung, spleen, and other tested organs. In all, tested organs donor cells undergo "licensing" for pathogenicity, consisting of vigorous increase in number and changes in expression levels of inflammation-related genes, monitored by both flow cytometry and microarray analysis. After reaching peak numbers, around day 3, the "licensed" donor cells migrate to the circulation and initiate inflammation in the HEL-expressing recipient eyes. Importantly, the kinetics of increase in number and of changes in gene expression by the donor cells were similar in lung, spleen, and other tested organs of the recipient mice. Furthermore, the total numbers of donor cells in the spleen at their peaks were 10- to 100-fold larger in the spleen than in the lung, contradicting the notion that the lung is the organ where "licensing" takes place.
Asunto(s)
Enfermedades Autoinmunes/inmunología , Autoinmunidad/inmunología , Activación de Linfocitos/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Traslado Adoptivo , Animales , Autoantígenos/inmunología , Modelos Animales de Enfermedad , Citometría de Flujo , Pulmón/inmunología , Ratones , Ratones Transgénicos , Muramidasa/inmunología , Bazo/inmunologíaRESUMEN
Synthetic oligonucleotides (ODNs) containing CpG motifs stimulate human plasmacytoid dendritic cells (pDCs) to produce type-1 interferons (IFNs) and proinflammatory cytokines. Previous studies demonstrated that interferon regulatory factors (IRFs) play a central role in mediating CpG-induced pDC activation. This work explores the inverse effects of IRF5 and IRF8 (also known as IFN consensus sequence-binding protein) on CpG-dependent gene expression in the human CAL-1 pDC cell line. This cell line shares many of the phenotypic and functional properties of freshly isolated human pDCs. Results from RNA interference and microarray studies indicate that IRF5 upregulates TLR9-driven gene expression whereas IRF8 downregulates the same genes. Several findings support the conclusion that IRF8 inhibits TLR9-dependent gene expression by directly blocking the activity of IRF5. First, the inhibitory activity of IRF8 is only observed when IRF5 is present. Second, proximity ligation analysis shows that IRF8 and IRF5 colocalize within the cytoplasm of resting human pDCs and cotranslocate to the nucleus after CpG stimulation. Taken together, these findings suggest that IRF5 and IRF8, two transcription factors with opposing functions, control TLR9 signaling in human pDCs.
Asunto(s)
Células Dendríticas/inmunología , Factores Reguladores del Interferón/inmunología , Receptor Toll-Like 9/inmunología , Receptor Toll-Like 9/metabolismo , Línea Celular , Citocinas/genética , Citocinas/inmunología , Células Dendríticas/efectos de los fármacos , Células Dendríticas/ultraestructura , Regulación hacia Abajo , Regulación de la Expresión Génica , Humanos , Análisis por Micromatrices , Oligodesoxirribonucleótidos/farmacología , Interferencia de ARN , Transducción de Señal , Receptor Toll-Like 9/genéticaRESUMEN
Systemic lupus is the prototypic human autoimmune disease. It is a kaleidoscope of autoreactivities, with clear indications of both a genetic and environmental basis. Indeed, it is a disease that can manifest in virtually every tissue and organ and can also be found spontaneously in a number of animal species, including dogs, cats and horses. Moreover, there are multiple murine models of lupus, the first of which, New Zealand Black (NZB) mice, were discovered in 1959. Despite an enormous effort from scientists in multiple disciplines, the etiology of lupus remains elusive and the introduction of new therapies has been disappointing. Fortunately, significant advances have occurred to help patients through the general principles of internal medicine, including antibiotics, dialysis, and of course use of steroids and immunosuppressive agents. However, the magic bullet has yet to be discovered. One of the major causes of morbidity in lupus remains lupus nephritis and there has been significant effort and encouragement in understanding the pathogenesis, renal histologic classification, and use of therapeutic protocols to induce and sustain remission of lupus nephritis. Indeed, the first use of evidence-based clinical trials in lupus was initiated by Dr. Alfred D. Steinberg at NIH in pioneering studies involving either oral or intravenous pulses of cyclophosphamide, azathioprine or corticosteroids alone and/or some combination. Cyclophosphamide intravenously proved to be superior and the use of cyclophosphamide in combination with methylprednisolone remained the standard protocol for the treatment of lupus nephritis for decades. Although alternative therapies have been introduced, including mycophenolate mofetil, the use of therapies first pioneered at NIH may still be considered standard of care in the appropriate indications. More targeted therapies are much desired. In this review we provide a comprehensive overview of lupus nephritis and the evolution of clinical treatments.
Asunto(s)
Nefritis Lúpica/terapia , Animales , Biopsia , Técnicas Histológicas/historia , Técnicas Histológicas/métodos , Historia del Siglo XX , Humanos , Nefritis Lúpica/diagnóstico , Nefritis Lúpica/historiaRESUMEN
Tumors persist by occupying immunosuppressive microenvironments that inhibit the activity of tumoricidal T and NK cells. Monocytic myeloid-derived suppressor cells (mMDSC) are an important component of this immunosuppressive milieu. We find that the suppressive activity of mMDSC isolated from cancer patients can be reversed by treatment with TLR7/8 agonists, which induce human mMDSC to differentiate into tumoricidal M1-like macrophages. In contrast, agonists targeting TLR1/2 cause mMDSC to mature into immunosuppressive M2-like macrophages. These two populations of macrophage are phenotypically and functionally discrete and differ in gene expression profile. The ability of TLR7/8 agonists to reverse mMDSC-mediated immune suppression suggests that they might be useful adjuncts for tumor immunotherapy.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Imidazoles/farmacología , Tolerancia Inmunológica/efectos de los fármacos , Monocitos/citología , Monocitos/efectos de los fármacos , Células Mieloides/citología , Células Mieloides/efectos de los fármacos , Receptores Toll-Like/agonistas , Humanos , Tolerancia Inmunológica/inmunología , Monocitos/inmunología , Células Mieloides/inmunología , Relación Estructura-Actividad , Receptores Toll-Like/inmunologíaRESUMEN
Synthetic oligodeoxynucleotides that can down-regulate cellular elements of the immune system have been developed and are being widely studied in preclinical models. These agents vary in sequence, mechanism of action, and cellular target(s) but share the ability to suppress a plethora of inflammatory responses. This work reviews the types of immunosuppressive oligodeoxynucleotide (Sup ODN) and compares their therapeutic activity against diseases characterized by pathologic levels of immune stimulation ranging from autoimmunity to septic shock to cancer (see graphical abstract). The mechanism(s) underlying the efficacy of Sup ODN and the influence size, sequence and nucleotide backbone on function are considered.
Asunto(s)
Inmunosupresores/química , Inmunosupresores/uso terapéutico , Oligodesoxirribonucleótidos/química , Oligodesoxirribonucleótidos/uso terapéutico , Animales , Secuencia de Bases , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Humanos , Inmunosupresores/farmacología , Factores Reguladores del Interferón/inmunología , Oligodesoxirribonucleótidos/farmacología , Receptor Toll-Like 9/inmunologíaRESUMEN
The primary goal of cancer immunotherapy is to elicit an immune response capable of eliminating the tumor. One approach toward accomplishing that goal uses general (rather than tumor-specific) immunomodulatory agents to boost the number and activity of pre-existing CTLs. We find that the intratumoral injection of polyguanosine (poly-G) oligonucleotides (ODN) has such an effect, boosting antitumor immunity and promoting tumor regression. The antitumor activity of poly-G ODN was mediated through CD8 T cells in a TLR9-independent manner. Mechanistically, poly-G ODN directly induced the phosphorylation of Lck (an essential element of the T cell-signaling pathway), thereby enhancing the production of IL-2 and CD8 T cell proliferation. These findings establish poly-G ODN as a novel type of cancer immunotherapy.
Asunto(s)
Antineoplásicos/metabolismo , Guanosina/fisiología , Interleucina-2/biosíntesis , Oligodesoxirribonucleótidos/biosíntesis , Oligodesoxirribonucleótidos/farmacología , Regulación hacia Arriba/inmunología , Secuencias de Aminoácidos/genética , Secuencias de Aminoácidos/inmunología , Animales , Línea Celular Tumoral , Islas de CpG/genética , Islas de CpG/inmunología , Guanosina/biosíntesis , Guanosina/genética , Humanos , Interleucina-2/genética , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Ratones Transgénicos , Oligodesoxirribonucleótidos/síntesis química , Fosforilación/efectos de los fármacos , Fosforilación/inmunología , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/metabolismo , Linfocitos T Citotóxicos/patología , Células Tumorales Cultivadas , Regulación hacia Arriba/genéticaRESUMEN
The interaction between TLRs and their cognate ligands triggers both the innate and adaptive immune systems, and thus can play a pivotal role in the defense against pathogen invasion. This work investigates the differentiation of naive CD4 cells into Th1 or Th17 phenotypes in mice treated with different TLR ligands. We use a model system in which naive transgenic cells specific to hen egg lysozyme are adoptively transferred into recipients that express hen egg lysozyme in the lens of the eye. The transferred naive T cells induce ocular inflammation only in recipients treated with TLR ligands. Treatment with LPS preferentially stimulated IL-17 production, whereas CpG oligodeoxynucleotide and polyinosinic:polycytidylic acid primarily stimulated Th1 cells. Peptidoglycan stimulated the two Th subpopulations equally. The preferential induction of Th1 or Th17 by the four ligands was detected in the spleen (where a major portion of the adoptively transferred cells homed) and in the eyes, where activated Th cells initiate inflammation. Analysis of the cytokines present in recipient mice suggests that Th1 induction is elicited by IL-12 and/or IFN-α, whereas Th17 generation is preferentially mediated by IL-6. Importantly, we show in this article that treatment with LPS selectively promoted in the recipient mice the generation of IL-6-producing activated B cells. An inverse correlation was found between the level of regulatory T cells and severity of inflammation induced by the donor cells. Taken together, our data show that specific TLR ligands differentially activate the immune system as evidenced by the generation of distinct Th phenotypes from naive CD4 cells.
Asunto(s)
Enfermedades Autoinmunes/inmunología , Células TH1/inmunología , Células Th17/inmunología , Receptores Toll-Like/metabolismo , Animales , Enfermedades Autoinmunes/metabolismo , Enfermedades Autoinmunes/patología , Pollos , Ligandos , Lipopolisacáridos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Muramidasa/genética , Peptidoglicano/metabolismo , Poli I-C/metabolismo , Células TH1/metabolismo , Células TH1/patología , Células Th17/metabolismo , Células Th17/patología , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 3/metabolismo , Receptor Toll-Like 4/metabolismo , Receptor Toll-Like 9/metabolismo , Receptores Toll-Like/fisiologíaRESUMEN
Synthetic oligodeoxynucleotides (ODNs) comprised of the immunosuppressive motif TTAGGG block TLR9 signaling, prevent STAT1 and STAT4 phosphorylation and attenuate a variety of inflammatory responses in vivo. In this study, we demonstrate that such suppressive ODN abrogate activation of cytosolic nucleic acid-sensing pathways. Pretreatment of dendritic cells and macrophages with the suppressive ODN-A151 abrogated type I IFN, TNF-α, and ISG induction in response to cytosolic dsDNA. In addition, A151 abrogated caspase-1-dependent IL-1ß and IL-18 maturation in dendritic cells stimulated with dsDNA and murine CMV. Inhibition was dependent on A151's phosphorothioate backbone, whereas substitution of the guanosine residues for adenosine negatively affected potency. A151 mediates these effects by binding to AIM2 in a manner that is competitive with immune-stimulatory DNA and as a consequence prevents AIM2 inflammasome complex formation. Collectively, these findings reveal a new route by which suppressive ODNs modulate the immune system and unveil novel applications for suppressive ODNs in the treatment of infectious and autoimmune diseases.
Asunto(s)
Inflamasomas/antagonistas & inhibidores , Proteínas Nucleares/metabolismo , Motivos de Nucleótidos , Oligodesoxirribonucleótidos/química , Oligodesoxirribonucleótidos/farmacología , Animales , Antiinflamatorios/química , Antiinflamatorios/farmacología , Línea Celular , Análisis por Conglomerados , Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/metabolismo , Citosol/metabolismo , ADN/metabolismo , Proteínas de Unión al ADN , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Inflamasomas/química , Inflamasomas/metabolismo , Ratones , Oligodesoxirribonucleótidos/metabolismo , Unión Proteica , Multimerización de Proteína/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Tionucleótidos/químicaRESUMEN
Alarmins are a group of structurally diverse host defense antimicrobial peptides that are important immune activators. In this article, we present a novel role for two potent alarmins, human ß-defensin 2 and 3 (HBD2 and 3), in promoting IFN-α production by human plasmacytoid dendritic cells. We demonstrate that HBD2 and 3 activate pDCs by enhancing the intracellular uptake of CpG and self DNA and promote DNA-induced IFN-α production in a TLR9-dependent manner. Both CpG and host DNA form aggregates that resemble DNA nets when combined with HBD2 and 3. Isothermal titration calorimetry studies to elucidate the nature of HBD3/CpG complexes demonstrate involvement of enthalpy-driven interactions, in addition to hydrophobic interactions, with the formation of complexes at a molar ratio of 2:1 defensin/CpG. The i.v. administration of HBD3/CpG complexes induced proinflammatory cytokines like IL-12, IFN-γ, IL-6, IFN-α, and IL-10 in serum, associated with an increased recruitment of APCs in the spleen. Subcutaneous injections of these complexes showed enhanced infiltration of inflammatory cells at the injection site, indicating a potential pathophysiological role for alarmin/DNA complexes in contributing to inflammation. Intraperitoneal immunization of HBD3/CpG complexes with OVA enhanced both cellular and humoral responses to OVA, compared with OVA/HBD3 or OVA/CPG alone, indicative of a much more potent adjuvant effect of the HBD3/CpG complexes. Thus, the ability of defensins to enhance cellular uptake of nucleic acids can lead to improved vaccine formulations by promoting their uptake by various cells, resulting in an enhanced immune response.
Asunto(s)
Células Dendríticas/metabolismo , Inflamación/inmunología , Interferón-alfa/biosíntesis , beta-Defensinas/metabolismo , Adyuvantes Inmunológicos/farmacología , Animales , Células Presentadoras de Antígenos/inmunología , Transporte Biológico , Células Cultivadas , Islas de CpG , ADN/metabolismo , Células Dendríticas/inmunología , Femenino , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Interferón-alfa/sangre , Interferón gamma/sangre , Interleucina-12/sangre , Interleucina-6/sangre , Ratones , Ratones Endogámicos C57BL , Ovalbúmina/inmunología , Receptor Toll-Like 9/metabolismoRESUMEN
Silicosis is an inflammatory lung disease induced by the inhalation of silica-containing dust particles. There is conflicting data on whether patients with silicosis are more susceptible to lung cancer induced by cigarette smoke. To examine this issue experimentally, a model was developed in which one of the most abundant and potent carcinogens present in cigarette smoke [4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK)] was administered to mice at the peak of silica-induced pulmonary inflammation. Results show that the incidence of lung tumors in silicotic mice treated with NNK was significantly increased compared with mice exposed to silica or NNK alone. Synthetic oligonucleotides (ODN) containing repetitive TTAGGG motifs can block pathologic inflammation. We therefore examined whether treatment with these suppressive (Sup) ODN could block silica-induced pulmonary inflammation and thereby reduce susceptibility to lung cancer. Results show that Sup (but not control) ODN inhibit pulmonary fibrosis and other inflammatory manifestations of chronic silicosis. Of greater import, Sup ODN reduced lung tumor incidence and multiplicity in silicotic mice exposed to NNK. These findings establish an experimental model for examining the role of silicotic inflammation in cancer susceptibility and demonstrate that Sup ODN represent a novel therapy for chronic silicosis.
Asunto(s)
Antiinflamatorios/farmacología , Susceptibilidad a Enfermedades , Neoplasias Pulmonares/etiología , Neoplasias Pulmonares/prevención & control , Oligodesoxirribonucleótidos/farmacología , Silicosis/complicaciones , Animales , Antiinflamatorios/administración & dosificación , Biomarcadores , Modelos Animales de Enfermedad , Femenino , Expresión Génica , Humanos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Neoplasias Pulmonares/patología , Ratones , Nitrosaminas/efectos adversos , Oligodesoxirribonucleótidos/administración & dosificación , Neumonía/complicaciones , Neumonía/tratamiento farmacológico , Neumonía/etiología , Neumonía/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Dióxido de Silicio/efectos adversos , Silicosis/patologíaRESUMEN
Synthetic oligonucleotides (ODN) expressing CpG motifs mimic the ability of bacterial DNA to trigger the innate immune system via TLR9. Plasmacytoid dendritic cells (pDCs) make a critical contribution to the ensuing immune response. This work examines the induction of antiviral (IFN-ß) and pro-inflammatory (IL-6) cytokines by CpG-stimulated human pDCs and the human CAL-1 pDC cell line. Results show that interferon regulatory factor-5 (IRF-5) and NF-κB p50 are key co-regulators of IFN-ß and IL-6 expression following TLR9-mediated activation of human pDCs. The nuclear accumulation of IRF-1 was also observed, but this was a late event that was dependant on type 1 IFN and unrelated to the initiation of gene expression. IRF-8 was identified as a novel negative regulator of gene activation in CpG-stimulated pDCs. As variants of IRF-5 and IRF-8 were recently found to correlate with susceptibility to certain autoimmune diseases, these findings are relevant to our understanding of the pharmacologic effects of "K" ODN and the role of TLR9 ligation under physiologic, pathologic, and therapeutic conditions.
Asunto(s)
Células Dendríticas/inmunología , Factores Reguladores del Interferón/inmunología , Interferón beta/biosíntesis , Interleucina-6/biosíntesis , Subunidad p50 de NF-kappa B/inmunología , Línea Celular , Células Dendríticas/metabolismo , Técnica del Anticuerpo Fluorescente , Regulación de la Expresión Génica/inmunología , Humanos , Immunoblotting , Inmunoprecipitación , Factores Reguladores del Interferón/metabolismo , Interferón beta/inmunología , Interleucina-6/inmunología , Subunidad p50 de NF-kappa B/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Receptor Toll-Like 9/inmunologíaRESUMEN
We generated a mouse model with a 162 nt AU-rich element (ARE) region deletion in the 3' untranslated region (3'UTR) of the interferon-gamma (IFN-γ) gene that results in chronic circulating serum IFN-γ levels. Mice homozygous for the ARE deletion (ARE-Del) (-/-) present both serologic and cellular abnormalities typical of patients with systemic lupus erythematosus (SLE). ARE-Del(-/-) mice display increased numbers of pDCs in bone marrow and spleen. Addition of IFN-γ to Flt3-ligand (Flt3L) treated in vitro bone marrow cultures results in a 2-fold increase in pDCs with concurrent increases in IRF8 expression. Marginal zone B (MZB) cells and marginal zone macrophages (MZMs) are absent in ARE-Del(-/-) mice. ARE-Del(+/-) mice retain both MZB cells and MZMs and develop no or mild autoimmunity. However, low dose clodronate treatment in ARE-Del(+/-) mice specifically eliminates MZMs and promotes anti-DNA antibody development and glomerulonephritis. Our findings demonstrate the consequences of a chronic IFN-γ milieu on B220(+) cell types and in particular the impact of MZB cell loss on MZM function in autoimmunity. Furthermore, similarities between disease states in ARE-Del(-/-) mice and SLE patients suggest that IFN-γ may not only be a product of SLE but may be critical for disease onset and progression.
Asunto(s)
Elementos Ricos en Adenilato y Uridilato/genética , Secuencia de Bases , Interferón gamma , Nefritis Lúpica/inmunología , Eliminación de Secuencia , Animales , Anticuerpos Antinucleares/inmunología , Linfocitos B/inmunología , Linfocitos B/patología , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/inmunología , Humanos , Factores Reguladores del Interferón/genética , Factores Reguladores del Interferón/inmunología , Interferón gamma/genética , Interferón gamma/inmunología , Nefritis Lúpica/genética , Macrófagos/inmunología , Macrófagos/patología , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Ratones , Ratones NoqueadosRESUMEN
Immunostimulatory CpG oligonucleotides (ODN) activate cells that express TLR9 and have been shown to improve the host's response to tumor Ags. Unfortunately, the immunosuppressive microenvironment that surrounds many cancers inhibits Ag-specific cellular responses and thus interferes with CpG-mediated immunotherapy. Myeloid-derived suppressor cells (MDSC) represent an important constituent of this immunosuppressive milieu. Large numbers of MDSC are present in and near tumor sites where they inhibit the activity of Ag-specific T and NK cells. Current studies indicate that the delivery of CpG ODN directly into the tumor bed reduces the immunosuppressive activity of monocytic (CD11b(+), Ly6G(-), Ly6C(high)) MDSC. Monocytic MDSC express TLR9 and respond to CpG stimulation by 1) losing their ability to suppress T cell function, 2) producing Th1 cytokines, and 3) differentiating into macrophages with tumoricidal capability. These findings provide insight into a novel mechanism by which CpG ODN contribute to tumor regression, and they support intratumoral injection as the optimal route for their delivery.
Asunto(s)
Tolerancia Inmunológica , Células Mieloides/inmunología , Neoplasias/inmunología , Oligodesoxirribonucleótidos/administración & dosificación , Oligodesoxirribonucleótidos/inmunología , Animales , Diferenciación Celular , Citocinas/biosíntesis , Células Dendríticas/inmunología , Inmunidad Innata/efectos de los fármacos , Inyecciones Intralesiones , Células Asesinas Naturales/inmunología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Linfocitos T/metabolismo , Linfocitos T Citotóxicos/inmunología , Receptor Toll-Like 9/biosíntesis , Microambiente Tumoral/inmunologíaRESUMEN
The interleukin (IL)-22R1 chain of the heterodimeric IL-22 receptor is not expressed on normal leukocytes, but this receptor is expressed on T cells from anaplastic lymphoma kinase-positive (ALK(+)) anaplastic large cell lymphoma (ALCL) patients. To investigate the consequences of aberrant expression of this receptor on lymphocytes, we generated transgenic mice that express IL-22R1 on lymphocytes. The health of these animals progressively deteriorated at 8 to 12 weeks of age, as they displayed respiratory distress, rough coat and sluggish movement, and subsequent lethality due to multiorgan inflammation. The IL-22R1 transgenic animals developed neutrophilia that correlated with increased levels of circulating IL-17 and granulocyte colony-stimulating factor. In addition, these mice had increased serum IL-22 levels, suggesting that T cells expressing IL-22R1 generate IL-22 in a positive autoregulatory loop. As a result of the mouse model findings, we analyzed circulating cytokine levels in ALK(+)ALCL patients and detected elevated levels of IL-22, IL-17, and IL-8 in untreated patient samples. Importantly, IL-22 and IL-17 were undetectable in all patients who were in complete remission after chemotherapy. This study documents a previously unknown role of IL-22R1 in inflammation and identifies the involvement of IL-22R1/IL-22 in ALK(+)ALCL.