RESUMEN
The acidic tumor microenvironment in melanoma drives immune evasion by up-regulating cyclic adenosine monophosphate (cAMP) in tumor-infiltrating monocytes. Here we show that the release of non-toxic concentrations of an adenylate cyclase (AC) inhibitor from poly(sarcosine)-block-poly(L-glutamic acid γ-benzyl ester) (polypept(o)id) copolymer micelles restores antitumor immunity. In combination with selective, non-therapeutic regulatory T cell depletion, AC inhibitor micelles achieve a complete remission of established B16-F10-OVA tumors. Single-cell sequencing of melanoma-infiltrating immune cells shows that AC inhibitor micelles reduce the number of anti-inflammatory myeloid cells and checkpoint receptor expression on T cells. AC inhibitor micelles thus represent an immunotherapeutic measure to counteract melanoma immune escape.
Asunto(s)
Inhibidores de Adenilato Ciclasa/farmacología , Adenilil Ciclasas/genética , Antineoplásicos/farmacología , AMP Cíclico/antagonistas & inhibidores , Melanoma Experimental/tratamiento farmacológico , Neoplasias Cutáneas/tratamiento farmacológico , Inhibidores de Adenilato Ciclasa/síntesis química , Adenilil Ciclasas/inmunología , Animales , Antineoplásicos/síntesis química , Compuestos de Bencilo/química , AMP Cíclico/inmunología , AMP Cíclico/metabolismo , Ésteres , Femenino , Expresión Génica , Humanos , Inmunidad Innata/efectos de los fármacos , Inyecciones Intralesiones , Proteína Antagonista del Receptor de Interleucina 1/genética , Proteína Antagonista del Receptor de Interleucina 1/inmunología , Melanoma Experimental/genética , Melanoma Experimental/inmunología , Melanoma Experimental/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Micelas , Células Mieloides/efectos de los fármacos , Células Mieloides/inmunología , Células Mieloides/patología , Péptidos/química , Ácido Poliglutámico/química , Receptores Inmunológicos/antagonistas & inhibidores , Receptores Inmunológicos/genética , Receptores Inmunológicos/inmunología , Sarcosina/análogos & derivados , Sarcosina/química , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/patología , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/patología , Carga Tumoral/efectos de los fármacos , Escape del Tumor/efectos de los fármacos , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/genética , Microambiente Tumoral/inmunologíaRESUMEN
PROBLEM: The cAMP pathway is involved in important biological processes including immune regulation and hormone signaling. At the feto-maternal unit, cAMP participates in placental function/physiology and the establishment of immunoendocrine networks. Low cAMP in male fetuses cord blood has been linked to poorer perinatal outcomes; however, cAMP placental content and its relationship with immune factors and fetal sex in an infectious condition have not been investigated. METHOD OF STUDY: Sex-dependent changes in cAMP content and its association with cytokines and antimicrobial peptides expression were studied in human placentas collected from normal pregnancies and with urinary tract infections (UTI). Radioimmunoassay was used to quantify cAMP in placental tissue, while immune markers expression was studied by qPCR. Additionally, cAMP effect on antimicrobial peptides expression was studied in cultured trophoblasts challenged with lipopolysaccharide, to mimic an infection. RESULTS: In UTI, placentas from female neonates had higher cAMP tissue content and increased expression of TNFA, IL1B, and IL10 than those from males, where IFNG was more elevated. While cAMP negatively correlated with maternal bacteriuria and IFNG, it positively correlated with the antimicrobial peptide S100A9 expression in a sex-specific fashion. In cultured trophoblasts, cAMP significantly stimulated ß-defensin-1 while reduced the lipopolysaccharide-dependent stimulatory effect on ß-defensin-2, ß-defensins-3, and S100A9. CONCLUSION: Our results showed higher cAMP content and defense cytokines expression in placentas associated with female neonates from pregnancies complicated by UTI. The associations between cAMP and bacteriuria/immune markers, together with cAMP's ability to differentially regulate placental antimicrobial peptides expression, suggest a dual modulatory role for cAMP in placental immunity.
Asunto(s)
AMP Cíclico/inmunología , Citocinas/inmunología , Placenta/inmunología , Complicaciones Infecciosas del Embarazo/inmunología , Infecciones Urinarias/inmunología , Estudios Transversales , AMP Cíclico/metabolismo , Femenino , Humanos , Recién Nacido , Masculino , Placenta/metabolismo , Embarazo , Complicaciones Infecciosas del Embarazo/metabolismo , Caracteres Sexuales , Infecciones Urinarias/metabolismoRESUMEN
Intradermal (ID) immunization is an attractive route of vaccination because it targets tissue rich in dendritic cells, has dose-sparing potential, and allows needle-free delivery. However, few adjuvants are effective, nonreactogenic, and compatible with needle-free delivery devices. In this study, we demonstrate that a combination adjuvant composed of cyclic-di-AMP (cdAMP) and the plant-derived nanoparticle adjuvant Nano-11 significantly enhanced the immune response to ID-injected vaccines in mice and pigs with minimal local reaction at the injection site. The cdAMP/Nano-11 combination adjuvant increased Ag uptake by lymph node-resident and migratory skin dendritic cell subpopulations, including Langerhans cells. ID immunization with cdAMP/Nano-11 expanded the population of germinal center B cells and follicular helper T cells in the draining lymph node and Ag-specific Th1 and Th17 cells in the spleen. It elicited an enhanced immune response with a significant increase of IgG1 and IgG2a responses in mice at a reduced dose compared with i.m. immunization. An increased IgG response was observed following needle-free ID immunization of pigs. Nano-11 and cdAMP demonstrated a strong synergistic interaction, as shown in the activation of mouse, human, and porcine APC, with increased expression of costimulatory molecules and secretion of TNF and IL-1ß. The combination adjuvant induced robust activation of both NF-κB and IFN regulatory factor signaling pathways and the NLRP3 inflammasome. We conclude that the combination of Nano-11 and cdAMP is a promising adjuvant for ID delivery of vaccines that supports a balanced immune response.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , AMP Cíclico/inmunología , Células Dendríticas/inmunología , Nanopartículas/administración & dosificación , Células TH1/inmunología , Animales , Células Cultivadas , Femenino , Humanos , Inmunidad Celular , Inmunidad Humoral , Inmunización , Mediadores de Inflamación/metabolismo , Inyecciones Intradérmicas , Ratones , Ratones Endogámicos BALB C , FN-kappa B/metabolismo , Plantas , Transducción de Señal , PorcinosRESUMEN
Nephritis remains the most common severe manifestation of systemic lupus erythematosus in which auto-antibodies mediate chronic inflammation and kidney damage. cAMP-phosphodiesterases regulate sodium excretion and inflammation in various tissues. How cAMP elevation can reduce systemic inflammation and suppress kidney inflammation and damage remains elusive. PDE4 signaling and cAMP metabolism were investigated along immune complex depositions in target tissues and kidney damage (histology). SLE disease progression is associated with changes in kidney PDE4 activity and expression. Moreover, lupus prone mice exhibit low kidney cAMP level which is associated to induction and relocation of nuclear and cytoskeleton PDE4 isoforms. Auto-antibodies-induced kidney damage was attested by mesangial proliferation and cellular infiltration. Interestingly, we reported that NCS 613 treatment decreases systemic auto-antibody secretion and their corresponding immune complex deposition in target tissues. Furthermore, NCS 613 is able to increase cAMP levels in the kidney; hence this compound rescues kidney PDE4 alterations in treated mice. NCS 613 overcomes disease progression in lupus prone mice by improving wellbeing and decreasing inflammation in treated mice. The PDE4 inhibitor, NCS 613, is a new anti-inflammatory compound that is believed to be a leading drug candidate for the treatment of inflammatory diseases such as lupus nephritis.
Asunto(s)
Adenina/análogos & derivados , Antiinflamatorios/uso terapéutico , Riñón/efectos de los fármacos , Nefritis Lúpica/tratamiento farmacológico , Inhibidores de Fosfodiesterasa 4/uso terapéutico , Adenina/farmacología , Adenina/uso terapéutico , Animales , Antiinflamatorios/farmacología , Complejo Antígeno-Anticuerpo/análisis , Complejo Antígeno-Anticuerpo/inmunología , AMP Cíclico/análisis , AMP Cíclico/inmunología , Femenino , Inflamación/tratamiento farmacológico , Inflamación/inmunología , Inflamación/patología , Riñón/inmunología , Riñón/patología , Lupus Eritematoso Sistémico/tratamiento farmacológico , Lupus Eritematoso Sistémico/inmunología , Lupus Eritematoso Sistémico/patología , Nefritis Lúpica/inmunología , Nefritis Lúpica/patología , Ratones Endogámicos MRL lpr , Inhibidores de Fosfodiesterasa 4/farmacologíaRESUMEN
Cyclic AMP (cAMP) is involved in many biological processes but little is known regarding its role in shaping immunity. Here we show that cAMP-PKA-CREB signaling (a pattern recognition receptor [PRR]-independent mechanism) regulates conventional type-2 Dendritic Cells (cDC2s) in mice and reprograms their Th17-inducing properties via repression of IRF4 and KLF4, transcription factors essential for cDC2-mediated Th2 induction. In mice, genetic loss of IRF4 phenocopies the effects of cAMP on Th17 induction and restoration of IRF4 prevents the cAMP effect. Moreover, curdlan, a PRR-dependent microbial product, activates CREB and represses IRF4 and KLF4, resulting in a pro-Th17 phenotype of cDC2s. These in vitro and in vivo results define a novel signaling pathway by which cDC2s display plasticity and provide a new molecular basis for the classification of novel cDC2 and cDC17 subsets. The findings also reveal that repressing IRF4 and KLF4 pathway can be harnessed for immuno-regulation.
Asunto(s)
Factores Reguladores del Interferón , Receptores de Reconocimiento de Patrones , Transducción de Señal/inmunología , Células Th17 , Células Th2 , Animales , Línea Celular Tumoral , AMP Cíclico/inmunología , AMP Cíclico/metabolismo , Citocinas , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Humanos , Factores Reguladores del Interferón/antagonistas & inhibidores , Factores Reguladores del Interferón/inmunología , Factores Reguladores del Interferón/metabolismo , Factor 4 Similar a Kruppel , Ratones , Receptores de Reconocimiento de Patrones/inmunología , Receptores de Reconocimiento de Patrones/metabolismo , Células Th17/inmunología , Células Th17/metabolismo , Células Th2/inmunología , Células Th2/metabolismoRESUMEN
Objectives: Regulatory T cells (Tregs) are frequently functionally impaired in patients with granulomatosis with polyangiitis (GPA). However, the mechanism underlying their impaired function is unknown. Here, we hypothesized that Treg dysfunction in GPA is due to altered microRNA (miRNA) expression. Methods: RNA isolated from FACS-sorted memory (M) Tregs (CD4+CD45RO+CD25+CD127-) of 8 healthy controls (HCs) and 8 GPA patients without treatment was subjected to miRNA microarray analysis. Five differentially expressed miRNAs were validated in a larger cohort by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). An miRNA target gene database search revealed targets that were tested with RT-qPCR in MTregs from patients and HCs. cAMP levels were measured using flow cytometry. Results: Microarray analysis revealed 19 differentially expressed miRNAs, of which miR-142-3p was confirmed to be significantly upregulated in MTregs from GPA patients compared to those from HCs (1.9-fold, p = 0.03). In vitro overexpression of miR-142-3p lowered the suppressive capacity of MTregs (2.1-fold, p = 0.03), and miR-142-3p expression correlated negatively with the suppressive capacity (rho = -0.446, p = 0.04). Overexpression of miR-142-3p significantly decreased cAMP levels (p = 0.02) and tended to decrease the mRNA levels of a predicted target gene, adenylate cyclase 9 (ADCY9; p = 0.06). In comparison to those from HCs, MTregs from GPA patients had lower ADCY9 mRNA levels (2-fold, p = 0.008) and produced significantly less cAMP after stimulation. Importantly, induction of cAMP production in miR-142-3p overexpressed MTregs by forskolin restored their suppressive function in vitro. Conclusion: Overexpression of miR-142-3p in MTregs from GPA patients might cause functional impairment by targeting ADCY9, which leads to the suppression of cAMP production.
Asunto(s)
Granulomatosis con Poliangitis/inmunología , MicroARNs/inmunología , Linfocitos T Reguladores/inmunología , Adenilil Ciclasas/genética , AMP Cíclico/inmunología , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Multiple sclerosis (MS) is a chronic demyelinating disease of the central nervous system (CNS) characterized by heterogeneous clinical symptoms including gradual muscle weakness, fatigue, and cognitive impairment. The disease course of MS can be classified into a relapsing-remitting (RR) phase defined by periods of neurological disabilities, and a progressive phase where neurological decline is persistent. Pathologically, MS is defined by a destructive immunological and neuro-degenerative interplay. Current treatments largely target the inflammatory processes and slow disease progression at best. Therefore, there is an urgent need to develop next-generation therapeutic strategies that target both neuroinflammatory and degenerative processes. It has been shown that elevating second messengers (cAMP and cGMP) is important for controlling inflammatory damage and inducing CNS repair. Phosphodiesterases (PDEs) have been studied extensively in a wide range of disorders as they breakdown these second messengers, rendering them crucial regulators. In this review, we provide an overview of the role of PDE inhibition in limiting pathological inflammation and stimulating regenerative processes in MS.
Asunto(s)
Esclerosis Múltiple , Inhibidores de Fosfodiesterasa/uso terapéutico , Hidrolasas Diéster Fosfóricas/inmunología , Sistemas de Mensajero Secundario , AMP Cíclico/inmunología , GMP Cíclico/inmunología , Humanos , Esclerosis Múltiple/tratamiento farmacológico , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/patología , Sistemas de Mensajero Secundario/efectos de los fármacos , Sistemas de Mensajero Secundario/inmunologíaRESUMEN
Malignant cells acquire physiological mechanisms of immunosuppression to escape immune surveillance. Strategies to counteract this suppression could help to improve adoptive immunotherapy regimen. The intracellular second messenger cyclic AMP (cAMP) acts as a potent immunosuppressive signaling molecule in T-cells and is up-regulated by multiple tumor-relevant suppressive factors including prostaglandin E2 (PGE2), adenosine and the functions of regulatory T-cells. Consequently, we aimed to abrogate cAMP signaling in human T-cells by ectopic overexpression of phosphodiesterase 4A (PDE4A). We could show that retroviral transduction of PDE4A into T-cells led to efficient degradation of cAMP in response to induction of adenylate cyclase. Retroviral transduction of PDE4A into CD4+ and CD8+ T-cells restored proliferation, cytokine secretion as well as cytotoxicity under immunosuppression by PGE2 and A2A-R agonists. PDE4A-transgenic T-cells were also partially protected from suppression by regulatory T-cells. Furthermore, PGE2-mediated upregulation of the inhibitory surface markers CD73 and CD94 on CD8+ T-cells was efficiently counteracted by PDE4A. Importantly, no differences in the functionality under non-suppressive conditions between PDE4A- and control-vector transduced T-cells were observed, indicating that PDE4A does not interfere with T-cell activation per se. Similarly, expression of surface markers associated with T-cell exhaustion were not influenced by PDE4A overexpression in long term cultures. Thus, we provide first in vitro evidence that PDE4A can be exploited as immune checkpoint inhibitor against multiple suppressive factors.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , AMP Cíclico/inmunología , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/inmunología , Regulación Enzimológica de la Expresión Génica/inmunología , Tolerancia Inmunológica , Linfocitos T Reguladores/inmunología , AMP Cíclico/genética , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/genética , Dinoprostona/genética , Dinoprostona/inmunología , Células HEK293 , HumanosRESUMEN
Therapeutic manipulation of regulatory T cells (Tregs) has been regarded as a promising approach for the treatment of immune disorders. However, a better understanding of the immunomodulatory mechanisms of Tregs and new safe and effective methods to improve the therapeutic effects of Tregs are highly desired. In this study, we have identified the key roles of a cAMP-adenosine positive feedback loop in the immunomodulatory function of Tregs. Adult male C57BL/6J mice were used for an experimental autoimmune uveitis (EAU) model, Tregs, and uveitogenic T cells (UTs). In established EAU, induced Tregs (iTregs) administration alleviated the inflammatory response. In vitro, iTregs inhibited UTs proliferation and inflammatory cytokine production. Mechanistically, cAMP is partially responsible for iTreg-mediated inhibition on UTs. Importantly, intracellular cAMP regulates CD39 expression and CD39-dependent adenosine production in iTregs, and cAMP directly participates in iTreg-derived adenosine production by a CD39 signaling-independent extracellular cAMP-adenosine pathway. Moreover, extracellular adenosine increases the intracellular cAMP level in Tregs. More importantly, increasing the cAMP level in iTregs before transfer improves their therapeutic efficacy in established EAU. Notably, the cAMP-adenosine loop exists in both iTregs and naturally occurring Tregs. These findings provide new insights into the immunosuppressive mechanisms of Tregs and suggest a new strategy for improving the therapeutic efficacy of Tregs in established autoimmune disease.
Asunto(s)
Adenosina/inmunología , AMP Cíclico/inmunología , Linfocitos T Reguladores/inmunología , Animales , Antígenos CD/inmunología , Apirasa/inmunología , Enfermedades Autoinmunes/inmunología , Femenino , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Uveítis/inmunologíaRESUMEN
Bacillus Calmette-Guerin (BCG) is a live attenuated vaccine against tuberculosis (TB) and remains the most commonly used vaccine worldwide. However, BCG has varied protective efficiency in adults and has safety concerns in immunocompromised population. Thus, effective vaccines are necessary for preventing the prevalence of TB. Cyclic di-AMP (c-di-AMP) is a bacterial second messenger which regulates various cellular processes and host immune response. Previous work found that c-di-AMP regulates bacterial physiological function, pathogenicity and host type I IFN response. In this study, we constructed a recombinant BCG (rBCG) by overexpressing DisA, the diadenylate cyclase of Mycobacterium tuberculosis (Mtb), and observed the physiological changes of rBCG-DisA. The immunological characteristics of rBCG-DisA were investigated on humoral and cellar immune responses in a mice infection model. Our study demonstrated that overexpression of DisA in BCG does not affect the growth but reduces the length of BCG. rBCG-DisA-immunized mice show similar humoral and cellar immune responses in BCG-immunized mice. After Mtb infection, the splenic lymphocytes from both BCG and rBCG-DisA-immunized mice produced more IFN-γ, IL-2, and IL-10 than the un-immunized (UN) mice, while the cytokine levels of the rBCG-DisA group increased significantly than those of the BCG group. The transcription of IFN-ß, IL-1ß and autophagy related genes (Atgs) were up-regulated in macrophages after treated with c-di-AMP or bacterial infection. The productions of IL-6 were increased after Mtb challenge, especially in the rBCG-DisA-immunized mice. Strikingly, H3K4me3, the epigenetic marker of innate immune memory, was found in both two immunized groups, and the rBCG-DisA group showed stronger expression of H3K4me3 than that of BCG. In addition, the pathological changes of rBCG-DisA immunized mice were similar to that of BCG-immunized mice. The bacterial burdens in the lungs and spleens of BCG- and rBCG-DisA-immunized mice were significantly decreased, but there was no significant difference between the two immunized groups. Together, these results suggested that compared to BCG, rBCG-DisA vaccination, induces stronger immune responses but did not provided additional protection against Mtb infection in this study, which may be related to the innate immunity memory. Hence, c-di-AMP is a promising immunomodulator for a further developed BCG as a better vaccine.
Asunto(s)
Adyuvantes Inmunológicos , Antígenos Bacterianos , Vacuna BCG , AMP Cíclico/inmunología , Inmunización , Tuberculosis , Animales , Antígenos Bacterianos/genética , Antígenos Bacterianos/inmunología , Vacuna BCG/genética , Vacuna BCG/inmunología , Vacuna BCG/farmacología , AMP Cíclico/genética , Citocinas/inmunología , Ratones , Células RAW 264.7 , Tuberculosis/genética , Tuberculosis/inmunología , Tuberculosis/patología , Tuberculosis/prevención & controlRESUMEN
Potentiation of neutrophil extracellular trap (NET) release is one mechanism by which antiphospholipid antibodies (aPL Abs) effect thrombotic events in patients with antiphospholipid syndrome (APS). Surface adenosine receptors trigger cyclic AMP (cAMP) formation in neutrophils, and this mechanism has been proposed to regulate NETosis in some contexts. Here we report that selective agonism of the adenosine A2A receptor (CGS21680) suppresses aPL Ab-mediated NETosis in protein kinase A-dependent fashion. CGS21680 also reduces thrombosis in the inferior vena cavae of both control mice and mice administered aPL Abs. The antithrombotic medication dipyridamole is known to potentiate adenosine signaling by increasing extracellular concentrations of adenosine and interfering with the breakdown of cAMP. Like CGS21680, dipyridamole suppresses aPL Ab-mediated NETosis via the adenosine A2A receptor and mitigates venous thrombosis in mice. In summary, these data suggest an anti-inflammatory therapeutic paradigm in APS, which may extend to thrombotic disease in the general population.
Asunto(s)
Agonistas del Receptor de Adenosina A2/farmacología , Adenosina/análogos & derivados , Síndrome Antifosfolípido/tratamiento farmacológico , Trampas Extracelulares/efectos de los fármacos , Neutrófilos/efectos de los fármacos , Fenetilaminas/farmacología , Trombosis de la Vena/tratamiento farmacológico , Adenosina/inmunología , Adenosina/metabolismo , Adenosina/farmacología , Animales , Anticuerpos Antifosfolípidos/sangre , Síndrome Antifosfolípido/genética , Síndrome Antifosfolípido/inmunología , Síndrome Antifosfolípido/patología , AMP Cíclico/inmunología , AMP Cíclico/metabolismo , Dipiridamol/farmacología , Modelos Animales de Enfermedad , Trampas Extracelulares/inmunología , Trampas Extracelulares/metabolismo , Fibrinolíticos/farmacología , Regulación de la Expresión Génica , Humanos , Inmunoglobulina G/sangre , Masculino , Ratones , Ratones Endogámicos C57BL , Neutrófilos/inmunología , Receptor de Adenosina A2A/genética , Receptor de Adenosina A2A/inmunología , Transducción de Señal , Vena Cava Inferior/efectos de los fármacos , Vena Cava Inferior/inmunología , Vena Cava Inferior/metabolismo , Trombosis de la Vena/genética , Trombosis de la Vena/inmunología , Trombosis de la Vena/patologíaRESUMEN
Cholera toxin (CT) is widely used as an effective adjuvant in experimental immunology for inducing mucosal immune responses; yet its mechanisms of adjuvant action remain incompletely defined. Here, we demonstrate that mice lacking NFκB, compared to wild-type (WT) mice, had a 90% reduction in their systemic and mucosal immune responses to oral immunization with a model protein antigen [Ovalbumin (OVA)] given together with CT. Further, NFκB-/- mouse dendritic cells (DCs) stimulated in vitro with CT showed reduced expression of MHCII and co-stimulatory molecules, such as CD80 and CD86, as well as of IL-1ß, and other pro-inflammatory cytokines compared to WT DCs. Using a human monocyte cell line THP1 with an NFκB activation reporter system, we show that CT induced NFκB signaling in human monocytes, and that inhibition of the cyclic AMP-protein kinase A (cAMP-PKA) pathway abrogated the activation and nuclear translocation of NFκB. In a human monocyte-CD4+ T cell co-culture system we further show that the strong Th17 response induced by CT treatment of monocytes was abolished by blocking the classical but not the alternative NFκB signaling pathway of monocytes. Our results indicate that activation of classical (canonical) NFκB pathway signaling in antigen-presenting cells (APCs) by CT is important for CT's adjuvant enhancement of Th17 responses. Similar findings were obtained using the almost completely detoxified mmCT mutant protein as adjuvant. Altogether, our results demonstrate that activation of the classical NFκB signal transduction pathway in APCs is important for the adjuvant action of both CT and mmCT.
Asunto(s)
Toxina del Cólera/inmunología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , FN-kappa B/metabolismo , Transducción de Señal/fisiología , Adyuvantes Inmunológicos/metabolismo , Animales , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/metabolismo , Antígenos/inmunología , AMP Cíclico/inmunología , Proteínas Quinasas Dependientes de AMP Cíclico/inmunología , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Femenino , Inmunidad Mucosa/inmunología , Ratones , Ratones Endogámicos C57BL , Monocitos/inmunología , Monocitos/metabolismo , FN-kappa B/inmunología , Ovalbúmina/inmunología , Transducción de Señal/inmunología , Células Th17/inmunología , Células Th17/metabolismoRESUMEN
Tregs play a fundamental role in immune tolerance via control of self-reactive effector T cells (Teffs). This function is dependent on maintenance of a high intracellular cAMP concentration. A number of microRNAs are implicated in the maintenance of Tregs. In this study, we demonstrate that peripheral immune tolerance is critically dependent on posttranscriptional repression of the cAMP-hydrolyzing enzyme phosphodiesterase-3b (Pde3b) by microRNA-142-5p (miR-142-5p). In this manner, miR-142-5p acts as an immunometabolic regulator of intracellular cAMP, controlling Treg suppressive function. Mir142 was associated with a super enhancer bound by the Treg lineage-determining transcription factor forkhead box P3 (FOXP3), and Treg-specific deletion of miR-142 in mice (TregΔ142) resulted in spontaneous, lethal, multisystem autoimmunity, despite preserved numbers of phenotypically normal Tregs. Pharmacological inhibition and genetic ablation of PDE3B prevented autoimmune disease and reversed the impaired suppressive function of Tregs in TregΔ142 animals. These findings reveal a critical molecular switch, specifying Treg function through the modulation of a highly conserved, cell-intrinsic metabolic pathway. Modulation of this pathway has direct relevance to the pathogenesis and treatment of autoimmunity and cancer.
Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/inmunología , Regulación Enzimológica de la Expresión Génica/inmunología , Tolerancia Inmunológica , MicroARNs/inmunología , Sistemas de Mensajero Secundario/inmunología , Linfocitos T Reguladores/inmunología , Animales , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/patología , AMP Cíclico/genética , AMP Cíclico/inmunología , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/genética , Regulación Enzimológica de la Expresión Génica/genética , Ratones , Ratones Transgénicos , MicroARNs/genética , Sistemas de Mensajero Secundario/genética , Linfocitos T Reguladores/patologíaRESUMEN
The ability of the immune system to discriminate self from non-self is essential for eradicating microbial pathogens but is also responsible for allograft rejection. Whether it is possible to selectively suppress alloresponses while maintaining anti-pathogen immunity remains unknown. We found that mice deficient in coronin 1, a regulator of naive T cell homeostasis, fully retained allografts while maintaining T cell-specific responses against microbial pathogens. Mechanistically, coronin 1-deficiency increased cyclic adenosine monophosphate (cAMP) concentrations to suppress allo-specific T cell responses. Costimulation induced on microbe-infected antigen presenting cells was able to overcome cAMP-mediated immunosuppression to maintain anti-pathogen immunity. In vivo pharmacological modulation of this pathway or a prior transfer of coronin 1-deficient T cells actively suppressed allograft rejection. These results define a coronin 1-dependent regulatory axis in T cells important for allograft rejection and suggest that modulation of this pathway may be a promising approach to achieve long-term acceptance of mismatched allografts.
Asunto(s)
Rechazo de Injerto/inmunología , Trasplante de Corazón , Infecciones/inmunología , Proteínas de Microfilamentos/metabolismo , Trasplante de Piel , Linfocitos T/inmunología , Aloinjertos/inmunología , Animales , Antígenos Bacterianos/inmunología , Antígenos Fúngicos/inmunología , Antígenos Virales/inmunología , Células Cultivadas , AMP Cíclico/inmunología , Supervivencia de Injerto , Homeostasis/genética , Humanos , Inmunidad , Terapia de Inmunosupresión , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Transducción de Señal , Tolerancia al TrasplanteRESUMEN
T cell immunoglobulin and mucin domain-3 (TIM-3) expression increases in exhausted T cells, which inhibits T cell function. TIM-3 expression is supposedly up-regulated in tumor-bearing individuals via chronic antigenic stimulation of T cells. Considering the immunosuppressive nature of the tumor microenvironment, we investigated whether tumor-secreted molecules might enhance TIM-3 expression in Jurkat T cells. We observed that TIM-3 expression was increased by the activation of prostaglandin (PG) E2 and cyclic AMP (cAMP) signaling pathways. Adenylate cyclase activation led to protein kinase A (PKA)-dependent upregulation of the TIM-3 minimal promoter region and of upstream conserved non-coding sequences. TIM-3 expression in Jurkat T cells was increased by the exposure to breast tumor cell-conditioned media partially through the interaction between PGE2 and its receptor, EP4. Our results propose that tumor-secreted molecules such as PGE2, which activates PKA and EPAC, may regulate TIM-3 expression in T cells.
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AMP Cíclico/inmunología , Regulación de la Expresión Génica/inmunología , Receptor 2 Celular del Virus de la Hepatitis A/inmunología , Neoplasias/inmunología , Sistemas de Mensajero Secundario/inmunología , Línea Celular Tumoral , Medios de Cultivo Condicionados/farmacología , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/inmunología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Dinoprostona/metabolismo , Dinoprostona/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Factores de Intercambio de Guanina Nucleótido/inmunología , Factores de Intercambio de Guanina Nucleótido/metabolismo , Receptor 2 Celular del Virus de la Hepatitis A/biosíntesis , Humanos , Células Jurkat , Células MCF-7 , Neoplasias/metabolismo , Sistemas de Mensajero Secundario/efectos de los fármacosRESUMEN
BACKGROUND: The potency of T regulatory (TREG) cells to inhibit T helper (Th)-driven immune cell responses has been linked to increased intracellular cyclic-AMP (cAMP) levels of TREG cells. In an ovalbumin (OVA)-driven allergic asthma mouse model, moderate aerobic exercise increases TREG cell function in a contact-dependent manner that leads to a significant reduction in chronic inflammation and restoration of lung function. However, the mechanism, whereby exercise increases TREG function, remains unknown and was the focus of these investigations. Exercise can communicate with TREG cells by their expression of ß2-adrenergic receptors (ß2-AR). Activation of these receptors results in an increase in intracellular levels of cyclic-AMP, potentially creating a potent inhibitor of Th cell responses. RESULTS: For the allergic asthma model, female wildtype BALB/c mice were challenged with OVA, and exercised (13.5 m/min for 45 min) 3×/week for 4 weeks. TREG cells were isolated from all mouse asthma/exercise groups, including ß2-AR-/- mice, to test suppressive function and intracellular cAMP levels. In these studies, cAMP levels were increased in TREG cells isolated from exercised mice. When ß2-AR expression was absent on TREG cells, cAMP levels were significantly decreased. Correlatively, their suppressive function was compromised. Next, TREG cells from all mouse groups were tested for suppressive function after treatment with either a pharmaceutical ß2-adrenergic agonist or an effector-specific cAMP analogue. These experiments showed TREG cell function was increased when treated with either a ß2-adrenergic agonist or effector-specific cAMP analogue. Finally, female wildtype BALB/c mice were antibody-depleted of CD25+CD4+ TREG cells (anti-CD25). Twenty-four hours after TREG depletion, either ß2-AR-/- or wildtype TREG cells were adoptively transferred. Recipient mice underwent the asthma/exercise protocols. ß2-AR-/- TREG cells isolated from these mice showed no increase in TREG function in response to moderate aerobic exercise. CONCLUSION: These studies offer a novel role for ß2-AR in regulating cAMP intracellular levels that can modify suppressive function in TREG cells.
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Asma/inmunología , Condicionamiento Físico Animal/métodos , Receptores Adrenérgicos beta 2/inmunología , Linfocitos T Reguladores/inmunología , Animales , Asma/metabolismo , AMP Cíclico/inmunología , AMP Cíclico/metabolismo , Modelos Animales de Enfermedad , Femenino , Espacio Intracelular/inmunología , Espacio Intracelular/metabolismo , Ratones Endogámicos BALB C , Ratones Transgénicos , Ovalbúmina/inmunología , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/metabolismo , Linfocitos T Reguladores/metabolismoRESUMEN
Cyclic adenosine monophosphate (cAMP) is critical in immune regulation, and its role in tuberculosis infection remains unclear. We determined the levels of cAMP in peripheral blood mononuclear cells (PBMC) from tuberculosis patients and the mechanisms for cAMP suppression of IFN-γ production. PBMC from tuberculosis patients contained significantly elevated cAMP than latent tuberculosis infected subjects (LTBI), with an inverse correlation with IFN-γ production. Consistent with this, the expression of cAMP response element binding protein (CREB), activating transcription factor (ATF)-2 and c-Jun were reduced in tuberculosis patients compared with LTBI. PKA type I specific cAMP analogs inhibited Mtb-stimulated IFN-g production by PBMC through suppression of Mtb-induced IFN-γ promoter binding activities of CREB, ATF-2, and c-Jun and also miR155, the target miRNA of these transcription factors. Neutralizing both IL-10 and TGF-ß1 or supplementation of IL-12 restored cAMP-suppressed IFN-g production. We conclude that increased cAMP inhibits IFN-g production through PKA type I pathway in tuberculosis infection.
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Proteína Quinasa Tipo I Dependiente de AMP Cíclico/inmunología , AMP Cíclico/inmunología , Interferón gamma/inmunología , Tuberculosis Latente/inmunología , Mycobacterium tuberculosis/inmunología , Linfocitos T/inmunología , Factor de Transcripción Activador 2/inmunología , Antígenos Bacterianos/inmunología , Humanos , Interleucina-10/inmunología , Leucocitos Mononucleares/inmunología , Regiones Promotoras Genéticas/inmunología , Unión Proteica/inmunología , Transducción de Señal/inmunologíaRESUMEN
BACKGROUND: Hydroxy-carboxylic acid receptor 2 (HCA2, also called GPR109A) belongs to the G protein-coupled receptor (GPCR) family and is found in humans, rats, mice, hamsters and guinea pigs, but there are almost no reports of this protein in other species. In this investigation, we speculated that AMP010014A09 (AMP+) is a homologue of GPR109A in swine. METHODS: To test this hypothesis, the following experiments were designed: monocytes isolated from the peripheral blood of swine were treated with LPS after pretreating with or without ß-hydroxybutyric acid (BHBA), and the levels of pro-inflammatory cytokines and inflammatory proteins were assessed. cAMP levels induced by Forskolin in swine testicular (ST) and IPEC-J2 cells were detected with or without BHBA treatment and following silencing or stable transfection of the AMP+ gene. RESULTS: AMP+ in swine exhibited a high level of homology with HM74A in humans and PUMA-G in mice. BHBA inhibited the LPS-induced secretion of the pro-inflammatory cytokines TNF-α, IL-6 and IL-1ß and the inflammatory protein COX-2 in monocytes of swine. BHBA suppressed the Forskolin-induced cAMP level increase in ST cells, but failed to inhibit the accumulation of cAMP after the AMP+ gene was silenced with shRNA by transfecting cells with the pGPU6-GFP-Neo-AMP+-sus-392 plasmid. BHBA had no effect on cAMP levels in IPEC-J2 cells, but significantly inhibited the increase in cAMP induced by Forskolin treatment following transfection of the AMP+ gene into IPEC-J2 cells by a lentivirus vector. CONCLUSION: Our results indicated that AMP+ encodes a G protein-coupled receptor in Sus scrofa that inhibits cAMP levels and mediates anti-inflammatory effects in swine monocytes.
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Ácido 3-Hidroxibutírico/farmacología , Antiinflamatorios no Esteroideos/farmacología , AMP Cíclico/inmunología , Monocitos/efectos de los fármacos , Receptores Acoplados a Proteínas G/inmunología , Receptores Nicotínicos/inmunología , Animales , Línea Celular , Colforsina/antagonistas & inhibidores , Colforsina/farmacología , AMP Cíclico/antagonistas & inhibidores , AMP Cíclico/metabolismo , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/inmunología , Expresión Génica , Intestinos/citología , Intestinos/efectos de los fármacos , Intestinos/inmunología , Lipopolisacáridos/antagonistas & inhibidores , Lipopolisacáridos/farmacología , Masculino , Ratones , Monocitos/citología , Monocitos/inmunología , Cultivo Primario de Células , Próstata/citología , Próstata/efectos de los fármacos , Próstata/inmunología , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/inmunología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/inmunología , Ratas , Receptores Acoplados a Proteínas G/genética , Receptores Nicotínicos/genética , Transducción de Señal , PorcinosRESUMEN
Studies showed that the use of cyclic adenosine monophosphate (cAMP) substitutes or intracellular cAMP activators increased intracellular cAMP level, causing anti-inflammatory effects. This study was to investigate the effects of pretreatment with meglumine cyclic adenylate (MCA), a compound of meglumine and cAMP, on systemic inflammation induced by lipopolysaccharide (LPS) in rats. Eighteen adult male Sprague-Dawley rats were randomly divided into 3 groups (n=6 each): control group (NS group), LPS group (LPS group) and LPS with MCA pretreatment group (MCA group). Systemic inflammation was induced with LPS 10 mg/kg injected via the femoral vein in LPS and MCA groups. In MCA group, MCA 2 mg/kg was injected via the femoral vein 20 min before LPS injection, and the equal volume of normal saline was given in NS and LPS groups at the same time. Three hours after LPS injection, the blood samples were taken from the abdominal aorta for determination of plasma concentrations of TNF-α, IL-1, IL-6, IL-10, cAMP by ELISA and NF-κBp65 expression by Western blotting. The experimental results showed that inflammatory and antiinflammatory indices were increased in LPS group compared to NS group; inflammatory indices were declined and anti-inflammatory indices were increased in MCA group relative to LPS group. Our study suggested that MCA pretreatment may attenuate LPS-induced systemic inflammation.
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Antiinflamatorios no Esteroideos/farmacología , AMP Cíclico/análogos & derivados , Meglumina/análogos & derivados , Síndrome de Respuesta Inflamatoria Sistémica/prevención & control , Animales , Biomarcadores/sangre , AMP Cíclico/sangre , AMP Cíclico/inmunología , AMP Cíclico/farmacología , Inyecciones Intravenosas , Interleucina-1/sangre , Interleucina-1/inmunología , Interleucina-10/sangre , Interleucina-10/inmunología , Interleucina-6/sangre , Interleucina-6/inmunología , Lipopolisacáridos , Masculino , Meglumina/farmacología , Ratas , Ratas Sprague-Dawley , Síndrome de Respuesta Inflamatoria Sistémica/sangre , Síndrome de Respuesta Inflamatoria Sistémica/inducido químicamente , Síndrome de Respuesta Inflamatoria Sistémica/inmunología , Factor de Transcripción ReIA/sangre , Factor de Transcripción ReIA/inmunología , Factor de Necrosis Tumoral alfa/sangre , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
The innate immune system is a critical component of host defence against microbial pathogens, but effective responses require an ability to distinguish between infectious and non-infectious insult to prevent inappropriate inflammation. Using the important obligate intracellular human pathogen Chlamydia trachomatis; an organism that causes significant immunopathology, we sought to determine critical host and pathogen factors that contribute to the induction of inflammasome activation. We assayed inflammasome activation by immunoblotting and ELISA to detect IL-1ß processing and LDH release to determine pyroptosis. Using primary murine bone marrow derived macrophages or human monocyte derived dendritic cells, infected with live or attenuated Chlamydia trachomatis we report that the live organism activates both canonical and non-canonical inflammasomes, but only canonical inflammasomes controlled IL-1ß processing which preceded pyroptosis. NADPH oxidase deficient macrophages were permissive to Chlamydia trachomatis replication and displayed elevated type-1 interferon and inflammasome activation. Conversely, attenuated, non-replicating Chlamydia trachomatis, primed but did not activate inflammasomes and stimulated reduced type-1 interferon responses. This suggested bacterial replication or metabolism as important factors that determine interferon responses and inflammasome activation. We identified STING but not cGAS as a central mediator of interferon regulated inflammasome activation. Interestingly, exogenous delivery of a Chlamydia trachomatis metabolite and STING ligand-cyclic di-AMP, recovered inflammasome activation to attenuated bacteria in a STING dependent manner thus indicating that a bacterial metabolite is a key factor initiating inflammasome activation through STING, independent of cGAS. These data suggest a potential mechanism of how the innate immune system can distinguish between infectious and non-infectious insult and instigate appropriate immune responses that could be therapeutically targeted.