RESUMEN
AIM: We aimed to test the hypothesis that a high-salt diet (HS) impairs NO signaling in kidney microvascular endothelial cells through a histone deacetylase 1 (HDAC1)-dependent mechanism. METHODS: Male Sprague Dawley rats were fed normal salt diet (NS; 0.49% NaCl) or HS (4% NaCl) for 2 weeks. NO signaling was assessed by measuring L-NAME induced vasoconstriction of the afferent arteriole using the blood perfused juxtamedullary nephron (JMN) preparation. In this preparation, kidneys were perfused with blood from a donor rat on a matching or different diet to that of the kidney donor. Kidney endothelial cells were isolated with magnetic activated cell sorting and HDAC1 activity was measured. RESULTS: We found HS-induced impaired NO signaling in the afferent arteriole. This was restored by inhibition of HDAC1 with MS-275. Consistent with these findings, HDAC1 activity was increased in kidney endothelial cells. We further found the loss of NO to be dependent upon the diet of the blood donor rather than the diet of the kidney donor and the plasma from HS-fed rats to be sufficient to induce impaired NO signaling. This indicates the presence of a humoral factor we termed plasma-derived endothelial dysfunction mediator (PDEM). Pretreatment with the antioxidants, PEG-SOD and PEG-catalase, as well as the NOS cofactor, tetrahydrobiopterin, restored NO signaling. CONCLUSION: We conclude that HS activates endothelial HDAC1 through PDEM leading to decreased NO signaling. This study provides novel insights into the molecular mechanisms by which a HS decreases renal microvascular endothelial NO signaling.
Asunto(s)
Histona Desacetilasa 1 , Riñón , Óxido Nítrico , Ratas Sprague-Dawley , Transducción de Señal , Cloruro de Sodio Dietético , Animales , Masculino , Histona Desacetilasa 1/metabolismo , Ratas , Óxido Nítrico/metabolismo , Riñón/metabolismo , Riñón/irrigación sanguínea , Riñón/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Células Endoteliales/metabolismo , Células Endoteliales/efectos de los fármacos , Microvasos/metabolismo , Microvasos/efectos de los fármacos , Endotelio Vascular/metabolismo , Endotelio Vascular/efectos de los fármacosRESUMEN
Type 1 diabetes (T1D) is an autoimmune disease culminating in the destruction of insulin-producing pancreatic cells. There is a need for the development of novel antigen-specific strategies to delay cell destruction, including combinatorial strategies that do not elicit systemic immunosuppression. Gamma-aminobutyric acid (GABA) is expressed by immune cells, ß-cells, and gut bacteria and is immunomodulatory. Glutamic-acid decarboxylase 65 (GAD65), which catalyzes GABA from glutamate, is a T1D autoantigen. To test the efficacy of combinatorial GABA treatment with or without GAD65-immunization to dampen autoimmune responses, we enrolled recent-onset children with T1D in a one-year clinical trial (ClinicalTrials.gov NCT02002130) and examined T cell responses. We isolated peripheral blood mononuclear cells and evaluated cytokine responses following polyclonal activation and GAD65 rechallenge. Both GABA alone and GABA/GAD65-alum treatment inhibited Th1 cytokine responses over the 12-month study with both polyclonal and GAD65 restimulation. We also investigated whether patients with HLA-DR3-DQ2 and HLA-DR4-DQ8, the two highest-risk human leukocyte antigen (HLA) haplotypes in T1D, exhibited differences in response to GABA alone and GABA/GAD65-alum. HLA-DR4-DQ8 patients possessed a Th1-skewed response compared to HLA-DR3-DQ2 patients. We show that GABA and GABA/GAD65-alum present an attractive immunomodulatory treatment for children with T1D and that HLA haplotypes should be considered.
RESUMEN
Aim: We aimed to identify new mechanisms by which a high salt diet (HS) decreases NO production in kidney microvascular endothelial cells. Specifically, we hypothesized HS impairs NO signaling through a histone deacetylase 1 (HDAC1)-dependent mechanism. Methods: Male Sprague Dawley rats were fed normal salt diet (NS; 0.49% NaCl) or high salt diet (4% NaCl) for two weeks. NO signaling was assessed by measuring L-NAME induced vasoconstriction of the afferent arteriole using the blood perfused juxtamedullary nephron (JMN) preparation. In this preparation, kidneys were perfused with blood from a donor rat on a matching or different diet to that of the kidney donor. Kidney endothelial cells were isolated with magnetic activated cell sorting and HDAC1 activity was measured. Results: We found that HS impaired NO signaling in the afferent arteriole. This was restored by inhibition of HDAC1 with MS-275. Consistent with these findings, HDAC1 activity was increased in kidney endothelial cells. We further found the loss of NO to be dependent upon the diet of the blood donor rather than the diet of the kidney donor and the plasma from HS fed rats to be sufficient to induce dysfunction suggesting a humoral factor, we termed Plasma Derived Endothelial-dysfunction Mediator (PDEM), mediates the endothelial dysfunction. The antioxidants, PEG-SOD and PEG-catalase, as well as the NOS cofactor, tetrahydrobiopterin, restored NO signaling. Conclusion: We conclude that HS activates endothelial HDAC1 through PDEM leading to decreased NO signaling. This study provides novel insights into the molecular mechanisms by which a HS decreases renal microvascular endothelial NO signaling.
RESUMEN
The coronavirus disease 2019 (COVID-19) pandemic has illustrated the critical need for effective prophylactic vaccination to prevent the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Intranasal vaccination is an attractive approach for preventing COVID-19 as the nasal mucosa is the site of initial SARS-CoV-2 entry and viral replication prior to aspiration into the lungs. We previously demonstrated that a single intranasal administration of a candidate adenovirus type 5-vectored vaccine encoding the receptor-binding domain of the SARS-CoV-2 spike protein (AdCOVID) induced robust immunity in both the airway mucosa and periphery, and completely protected K18-hACE2 mice from lethal SARS-CoV-2 challenge. Here we show that a single intranasal administration of AdCOVID limits viral replication in the nasal cavity of K18-hACE2 mice. AdCOVID also induces sterilizing immunity in the lungs of mice as reflected by the absence of infectious virus. Finally, AdCOVID prevents SARS-CoV-2 induced pathological damage in the lungs of mice. These data show that AdCOVID not only limits viral replication in the respiratory tract, but it also prevents virus-induced inflammation and immunopathology following SARS-CoV-2 infection.
Asunto(s)
Vacunas contra la COVID-19 , COVID-19 , SARS-CoV-2 , Animales , Humanos , Ratones , Administración Intranasal , Anticuerpos Antivirales , COVID-19/prevención & control , Pulmón , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus , Vacunas Virales/administración & dosificación , Vacunas contra la COVID-19/administración & dosificaciónRESUMEN
CD8+ T cell responses to pulmonary challenges are primed by lung migratory dendritic cells (mDCs), which capture antigens in the lungs and migrate to the lung-draining mediastinal lymph node (med-LN) to activate T cells. The lungs and the spleen are not connected by the lymphatic vasculature. Thus, the current paradigm suggests that, in response to respiratory virus infections that are restricted to the respiratory tract, priming of T cell responses by lung mDCs takes place entirely in the med-LN. Our results challenge this "LN-centric" paradigm by demonstrating that, during influenza virus infection, lung mDCs egress the med-LN and traffic to the spleen, where they prime influenza-specific CD8+ T cells. CD8+ T cells primed in the spleen are transcriptionally distinct and have enhanced ability to differentiate into long-lived memory cells compared with med-LNprimed counterparts. Thus, our data identify a lung mDC trafficking pathway that connects the lungs with the spleen.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Factor Nuclear 1-alfa del Hepatocito/inmunología , Pulmón/inmunología , Infecciones por Orthomyxoviridae/inmunología , Bazo/inmunología , Animales , Movimiento Celular/inmunología , Células Dendríticas/inmunología , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células Precursoras de Linfocitos T/inmunologíaRESUMEN
The coronavirus disease 2019 (COVID-19) pandemic has highlighted the urgent need for effective prophylactic vaccination to prevent the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Intranasal vaccination is an attractive strategy to prevent COVID-19 as the nasal mucosa represents the first-line barrier to SARS-CoV-2 entry. The current intramuscular vaccines elicit systemic immunity but not necessarily high-level mucosal immunity. Here, we tested a single intranasal dose of our candidate adenovirus type 5-vectored vaccine encoding the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein (AdCOVID) in inbred, outbred, and transgenic mice. A single intranasal vaccination with AdCOVID elicited a strong and focused immune response against RBD through the induction of mucosal IgA in the respiratory tract, serum neutralizing antibodies, and CD4+ and CD8+ T cells with a Th1-like cytokine expression profile. A single AdCOVID dose resulted in immunity that was sustained for over six months. Moreover, a single intranasal dose completely protected K18-hACE2 mice from lethal SARS-CoV-2 challenge, preventing weight loss and mortality. These data show that AdCOVID promotes concomitant systemic and mucosal immunity and represents a promising vaccine candidate.
RESUMEN
The coronavirus disease 2019 (COVID-19) pandemic has highlighted the urgent need for effective preventive vaccination to reduce burden and spread of severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2) in humans. Intranasal vaccination is an attractive strategy to prevent COVID-19 as the nasal mucosa represents the first-line barrier to SARS-CoV-2 entry before viral spread to the lung. Although SARS-CoV-2 vaccine development is rapidly progressing, the current intramuscular vaccines are designed to elicit systemic immunity without conferring mucosal immunity. Here, we show that AdCOVID, an intranasal adenovirus type 5 (Ad5)-vectored vaccine encoding the receptor binding domain (RBD) of the SARS-CoV-2 spike protein, elicits a strong and focused immune response against RBD through the induction of mucosal IgA, serum neutralizing antibodies and CD4+ and CD8+ T cells with a Th1-like cytokine expression profile. Therefore, AdCOVID, which promotes concomitant systemic and local mucosal immunity, represents a promising COVID-19 vaccine candidate.
RESUMEN
NAD, a key co-enzyme required for cell metabolism, is synthesized via two pathways in most organisms. Since schistosomes apparently lack enzymes required for de novo NAD biosynthesis, we evaluated whether these parasites, which infect >200 million people worldwide, maintain NAD homeostasis via the NAD salvage biosynthetic pathway. We found that intracellular NAD levels decline in schistosomes treated with drugs that block production of nicotinamide or nicotinamide mononucleotide-known NAD precursors in the non-deamidating salvage pathway. Moreover, in vitro inhibition of the NAD salvage pathway in schistosomes impaired egg production, disrupted the outer membranes of both immature and mature parasites and caused loss of mobility and death. Inhibiting the NAD salvage pathway in schistosome-infected mice significantly decreased NAD levels in adult parasites, which correlated with reduced egg production, fewer liver granulomas and parasite death. Thus, schistosomes, unlike their mammalian hosts, appear limited to one metabolic pathway to maintain NAD-dependent metabolic processes.
Asunto(s)
Interacciones Huésped-Parásitos/fisiología , NAD/metabolismo , Schistosoma mansoni/fisiología , Esquistosomiasis mansoni/metabolismo , Animales , Femenino , Ratones , Reproducción/fisiología , Esquistosomiasis mansoni/patologíaRESUMEN
Although B cells expressing the IFNγR or the IFNγ-inducible transcription factor T-bet promote autoimmunity in Systemic Lupus Erythematosus (SLE)-prone mouse models, the role for IFNγ signaling in human antibody responses is unknown. We show that elevated levels of IFNγ in SLE patients correlate with expansion of the T-bet expressing IgDnegCD27negCD11c+CXCR5neg (DN2) pre-antibody secreting cell (pre-ASC) subset. We demonstrate that naïve B cells form T-bethi pre-ASCs following stimulation with either Th1 cells or with IFNγ, IL-2, anti-Ig and TLR7/8 ligand and that IL-21 dependent ASC formation is significantly enhanced by IFNγ or IFNγ-producing T cells. IFNγ promotes ASC development by synergizing with IL-2 and TLR7/8 ligands to induce genome-wide epigenetic reprogramming of B cells, which results in increased chromatin accessibility surrounding IRF4 and BLIMP1 binding motifs and epigenetic remodeling of IL21R and PRDM1 loci. Finally, we show that IFNγ signals poise B cells to differentiate by increasing their responsiveness to IL-21.
Asunto(s)
Subgrupos de Linfocitos B/fisiología , Diferenciación Celular , Epigénesis Genética , Interferón gamma/metabolismo , Interleucinas/metabolismo , Receptor Toll-Like 7/metabolismo , Receptor Toll-Like 8/metabolismo , Subgrupos de Linfocitos B/química , Subgrupos de Linfocitos B/efectos de los fármacos , Redes Reguladoras de Genes , Humanos , Lupus Eritematoso Sistémico/patología , Proteínas de Dominio T Box/análisisRESUMEN
In this study, we investigated the role of CD38 in a pristane-induced murine model of lupus. CD38-deficient (Cd38-/-) but not ART2-deficient (Art2-/-) mice developed less severe lupus compared to wild type (WT) mice, and their protective phenotype consisted of (i) decreased IFN-I-stimulated gene expression, (ii) decreased numbers of peritoneal CCR2hiLy6Chi inflammatory monocytes, TNF-α-producing Ly6G+ neutrophils and Ly6Clo monocytes/macrophages, (iii) decreased production of anti-single-stranded DNA and anti-nRNP autoantibodies, and (iv) ameliorated glomerulonephritis. Cd38-/- pristane-elicited peritoneal exudate cells had defective CCL2 and TNF-α secretion following TLR7 stimulation. However, Tnf-α and Cxcl12 gene expression in Cd38-/- bone marrow (BM) cells was intact, suggesting a CD38-independent TLR7/TNF-α/CXCL12 axis in the BM. Chemotactic responses of Cd38-/- Ly6Chi monocytes and Ly6G+ neutrophils were not impaired. However, Cd38-/- Ly6Chi monocytes and Ly6Clo monocytes/macrophages had defective apoptosis-mediated cell death. Importantly, mice lacking the cation channel TRPM2 (Trpm2-/-) exhibited very similar protection, with decreased numbers of PECs, and apoptotic Ly6Chi monocytes and Ly6Clo monocytes/macrophages compared to WT mice. These findings reveal a new role for CD38 in promoting aberrant inflammation and lupus-like autoimmunity via an apoptosis-driven mechanism. Furthermore, given the implications of CD38 in the activation of TRPM2, our data suggest that CD38 modulation of pristane-induced apoptosis is TRPM2-dependent.
Asunto(s)
ADP-Ribosil Ciclasa 1/metabolismo , Apoptosis , Inmunosupresores/farmacología , Lupus Eritematoso Cutáneo/inducido químicamente , Lupus Eritematoso Cutáneo/patología , Glicoproteínas de Membrana/metabolismo , Canales Catiónicos TRPM/metabolismo , Terpenos/farmacología , ADP Ribosa Transferasas/deficiencia , ADP Ribosa Transferasas/metabolismo , ADP-Ribosil Ciclasa 1/deficiencia , Animales , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Factores Inmunológicos/metabolismo , Leucocitos/inmunología , Glicoproteínas de Membrana/deficiencia , RatonesRESUMEN
Interleukin 2 (IL-2) promotes Foxp3+ regulatory T (Treg) cell responses, but inhibits T follicular helper (TFH) cell development. However, it is not clear how IL-2 affects T follicular regulatory (TFR) cells, a cell type with properties of both Treg and TFH cells. Using an influenza infection model, we found that high IL-2 concentrations at the peak of the infection prevented TFR cell development by a Blimp-1-dependent mechanism. However, once the immune response resolved, some Treg cells downregulated CD25, upregulated Bcl-6 and differentiated into TFR cells, which then migrated into the B cell follicles to prevent the expansion of self-reactive B cell clones. Thus, unlike its effects on conventional Treg cells, IL-2 inhibits TFR cell responses.
Asunto(s)
Interleucina-2/farmacología , Infecciones por Orthomyxoviridae/inmunología , Orthomyxoviridae/inmunología , Linfocitos T Colaboradores-Inductores/efectos de los fármacos , Linfocitos T Reguladores/efectos de los fármacos , Animales , Movimiento Celular/genética , Movimiento Celular/inmunología , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/inmunología , Factores de Transcripción Forkhead/metabolismo , Perfilación de la Expresión Génica/métodos , Interacciones Huésped-Patógeno/efectos de los fármacos , Interacciones Huésped-Patógeno/inmunología , Interleucina-2/administración & dosificación , Interleucina-2/metabolismo , Subunidad alfa del Receptor de Interleucina-2/genética , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Orthomyxoviridae/fisiología , Infecciones por Orthomyxoviridae/metabolismo , Infecciones por Orthomyxoviridae/virología , Factor 1 de Unión al Dominio 1 de Regulación Positiva , Proteínas Proto-Oncogénicas c-bcl-6/genética , Proteínas Proto-Oncogénicas c-bcl-6/metabolismo , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T Colaboradores-Inductores/metabolismo , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/inmunología , Factores de Transcripción/metabolismoRESUMEN
SmNACE is a NAD catabolizing enzyme expressed on the outer tegument of S. mansoni, a human parasite that is one of the major agents of the neglected tropical disease schistosomiasis. Recently, we identified aroylhydrazone derivatives capable of inhibiting the recombinant form of the enzyme with variable potency (IC50 ranging from 88 µM to 33 nM). In the present study, we investigated the mechanism of action of the least potent micromolar inhibitor (compound 1) and the most potent nanomolar inhibitor (compound 2) in the series on both the recombinant and native SmNACE enzymes. Using mass spectroscopy, spectrophotometry, and activity assays under different experimental conditions, we demonstrated that the >3 log gain in potency against recombinant SmNACE by this class of compounds is dependent on the formation of a coordination complex with metal cations, such as Ni(II), Zn(II), and Fe(II), that are loaded on the protein surface. Testing the compounds on live parasites, we observed that only the weak micromolar compound 1 was active on the native enzyme. We showed that S. mansoni effectively sequesters the metal from the coordination complex, resulting in the loss of inhibitory activity of the potent nanomolar compound 2. Importantly, the modeling of the transition complex between Zn(II) and compound 2 enabled the discovery of a new metal-independent aroylhydrazone analogue, which is now the most potent and selective inhibitor of native SmNACE known.
Asunto(s)
Complejos de Coordinación/farmacología , Metales/metabolismo , Schistosoma mansoni/enzimología , Animales , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Concentración 50 Inhibidora , Estructura Molecular , Schistosoma mansoni/metabolismo , Zinc/químicaRESUMEN
Coordinate regulation of PARP-1 and -2 and PARG is required for cellular responses to genotoxic stress. While PARP-1 and -2 are regulated by DNA breaks and covalent modifications, mechanisms of PARG regulation are poorly understood. We report here discovery of a PARG regulatory segment far removed linearly from residues involved in catalysis. Expression and analysis of human PARG segments identified a minimal catalytically active C-terminal PARG (hPARG59) containing a 16-residue N-terminal mitochondrial targeting sequence (MTS). Deletion analysis and site-directed mutagenesis revealed that the MTS, specifically hydrophobic residues L473 and L474, was required for PARG activity. This region of PARG was termed the "regulatory segment/MTS" (REG/MTS). The overall alpha-helical composition of hPARG59, determined by circular dichroism (CD), was unaffected by mutation of the REG/MTS leucine residues, suggesting that activity loss was not due to incorrect protein folding. REG/MTS was predicted to be in a loop conformation because the CD spectra of mutant Delta1-16 lacking the REG/MTS showed a higher alpha-helical content than hPARG59, indicating a secondary structure other than alpha-helix for this segment. Deletion of the REG/MTS from full-length hPARG111 also resulted in a complete loss of activity, indicating that all PARG isoforms are subject to regulation at this site. The presence of the REG/MTS raises the possibility that PARG activity is regulated by interactions of PARP-1 and -2 and other proteins at this site, raises interesting questions concerning mitochondrial PARG because MTS residues are often removed after transport, and offers a potentially novel site for drug targeting of PARG.