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Although hyponatremia and salt wasting are common in patients with HIV/AIDS, the understanding of their contributing factors is limited. HIV viral protein R (Vpr) contributes to HIV-associated nephropathy. To investigate the effects of Vpr on the distal tubules and on the expression level of the Slc12a3 gene, encoding the sodium-chloride cotransporter (which is responsible for sodium reabsorption in distal nephron segments), single-nucleus RNA sequencing was performed on kidney cortices from three wild-type (WT) and three Vpr transgenic (Vpr Tg) mice. The results show that the percentage of distal convoluted tubule (DCT) cells was significantly lower in Vpr Tg mice compared with WT mice (P < 0.05); in Vpr Tg mice, Slc12a3 expression was not significantly different in DCT cells. The Pvalb+ DCT1 subcluster had fewer cells in Vpr Tg mice compared with WT mice (P < 0.01). Immunohistochemistry revealed fewer Slc12a3+Pvalb+ DCT1 segments in Vpr Tg mice. Differential gene expression analysis between Vpr Tg and WT samples in the DCT cluster showed down-regulation of the Ier3 gene, which is an inhibitor of apoptosis. The in vitro knockdown of Ier3 by siRNA transfection induced apoptosis in mouse DCT cells. These observations suggest that the salt-wasting effect of Vpr in Vpr Tg mice is likely mediated by Ier3 down-regulation in DCT1 cells and loss of Slc12a3+Pvalb+ DCT1 segments.
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Neutrophils play important roles in inflammatory airway diseases. In this study, we assessed whether apolipoprotein A-I modifies neutrophil heterogeneity as part of the mechanism by which it attenuates acute airway inflammation. Neutrophilic airway inflammation was induced by daily intranasal administration of LPS plus house dust mite (LPS+HDM) to Apoa1-/- and Apoa1+/+ mice for 3 d. Single-cell RNA sequencing was performed on cells recovered from bronchoalveolar lavage fluid on day 4. Unsupervised profiling identified 10 clusters of neutrophils in bronchoalveolar lavage fluid from Apoa1-/- and Apoa1+/+ mice. LPS+HDM-challenged Apoa1-/- mice had an increased proportion of the Neu4 neutrophil cluster that expressed S100a8, S100a9, and Mmp8 and had high maturation, aggregation, and TLR4 binding scores. There was also an increase in the Neu6 cluster of immature neutrophils, whereas neutrophil clusters expressing IFN-stimulated genes were decreased. An unsupervised trajectory analysis showed that Neu4 represented a distinct lineage in Apoa1-/- mice. LPS+HDM-challenged Apoa1-/- mice also had an increased proportion of recruited airspace macrophages, which was associated with a reciprocal reduction in resident airspace macrophages. Increased expression of a common set of proinflammatory genes, S100a8, S100a9, and Lcn2, was present in all neutrophils and airspace macrophages from LPS+HDM-challenged Apoa1-/- mice. These findings show that Apoa1-/- mice have increases in specific neutrophil and macrophage clusters in the lung during acute inflammation mediated by LPS+HDM, as well as enhanced expression of a common set of proinflammatory genes. This suggests that modifications in neutrophil and macrophage heterogeneity contribute to the mechanism by which apolipoprotein A-I attenuates acute airway inflammation.
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Apolipoproteína A-I , Ratones Noqueados , Neutrófilos , Neumonía , Animales , Ratones , Neutrófilos/inmunología , Neumonía/inmunología , Neumonía/genética , Apolipoproteína A-I/genética , Ratones Endogámicos C57BL , Lipopolisacáridos/inmunología , Líquido del Lavado Bronquioalveolar/inmunología , Líquido del Lavado Bronquioalveolar/citología , Pulmón/inmunología , Pulmón/patología , Calgranulina A , Calgranulina BRESUMEN
The neuroinflammatory process in synucleinopathies of the aging population such as Parkinson's disease (PD) and dementia with Lewy bodies (DLB) involves microglial activation as well as infiltration of the CNS by T cells and natural killer T cells (NKTs). To evaluate the potential of targeting NKT cells to modulate neuroinflammation, we treated α-syn transgenic (tg) mice (e.g.: Thy1 promoter line 61) with an antibody against CD1d, which is a glycoprotein expressed in antigen presenting cells (APCs). CD1d-presented lipid antigens activate NKT cells through the interaction with T cell receptor in NKTs, resulting in the production of cytokines. Thus, we hypothesized that blocking the APC-NKT interaction with an anti-CD1d antibody might reduce neuroinflammation and neurodegeneration in models of DLB/PD. Treatment with the anti-CD1d antibody did not have effects on CD3 (T cells), slightly decreased CD4 and increased CD8 lymphocytes in the mice. Moreover, double labeling studies showed that compared to control (IgG) treated α-syn tg mice, treatment with anti-CD1d decreased numbers of CD3/interferon γ (IFN γ)-positive cells, consistent with NKTs. Further double labeling studies showed that CD1d-positive cells co-localized with the astrocytes marker GFAP and that anti-CD1d antibody reduced this effect. While in control α-syn tg mice CD3 positive cells were near astrocytes, this was modified by the treatment with the CD1d antibody. By qPCR, levels of IFN γ, CCL4, and interleukin-6 were increased in the IgG treated α-syn tg mice. Treatment with CD1d antibody blunted this cytokine response that was associated with reduced astrocytosis and microgliosis in the CNS of the α-syn tg mice treated with CD1d antibody. Flow cytometric analysis of immune cells in α-syn tg mice revealed that CD1d-tet + T cells were also increased in the spleen of α-syn tg mice, which treatment with the CD1d antibody reduced. Reduced neuroinflammation in the anti-CD1d-treated α-syn tg mice was associated with amelioration of neurodegenerative pathology. These results suggest that reducing infiltration of NKT cells with an antibody against CD1d might be a potential therapeutical approach for DLB/PD.
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Enfermedad de Parkinson , alfa-Sinucleína , Ratones , Animales , alfa-Sinucleína/genética , Cuerpos de Lewy/patología , Enfermedades Neuroinflamatorias , Enfermedad de Parkinson/patología , Ratones Transgénicos , Inmunoterapia/métodos , Citocinas , Inmunoglobulina GRESUMEN
The transplantation of gene-modified autologous hematopoietic stem and progenitor cells (HSPCs) offers a promising therapeutic approach for hematological and immunological disorders. However, this strategy is often limited by the toxicities associated with traditional conditioning regimens. Antibody-based conditioning strategies targeting cKIT and CD45 antigens have shown potential in mitigating these toxicities, but their long-term safety and efficacy in clinical settings require further validation. In this study, we investigate the thrombopoietin (TPO) receptor, cMPL, as a novel target for conditioning protocols. We demonstrate that high surface expression of cMPL is a hallmark feature of long-term repopulating hematopoietic stem cells (LT-HSCs) within the adult human CD34+ HSPC subset. Targeting the cMPL receptor facilitates the separation of human LT-HSCs from mature progenitors, a delineation not achievable with cKIT. Leveraging this finding, we developed a cMPL-targeting immunotoxin, demonstrating its ability to selectively deplete host cMPLhigh LT-HSCs with a favorable safety profile and rapid clearance within 24 hours post-infusion in rhesus macaques. These findings present significant potential to advance our understanding of human hematopoiesis and enhance the therapeutic outcomes of ex vivo autologous HSPC gene therapies.
RESUMEN
Generally, fasting and refeeding confer anti- and proinflammatory effects, respectively. In humans, these caloric-load interventions function, in part, via regulation of CD4+ T cell biology. However, mechanisms orchestrating this regulation remain incomplete. We employed integrative bioinformatics of RNA sequencing and high-performance liquid chromatography-mass spectrometry data to measure serum metabolites and gene expression of peripheral blood mononuclear cells isolated from fasting and refeeding in volunteers to identify nutrient-load metabolite-driven immunoregulation. Propionate, a short chain fatty acid (SCFA), and the SCFA-sensing G protein-coupled receptor 43 (ffar2) were coordinately and inversely regulated by fasting and refeeding. Propionate and free fatty acid receptor agonists decreased interferon-γ and interleukin-17 and significantly blunted histone deacetylase activity in CD4+ T cells. Furthermore, propionate blunted nuclear factor κB activity and diminished interleukin-6 release. In parallel, propionate reduced phosphorylation of canonical T helper 1 (TH1) and TH17 regulators, STAT1 and STAT3, respectively. Conversely, knockdown of free fatty acid receptors significantly attenuated the anti-inflammatory role of propionate. Interestingly, propionate recapitulated the blunting of CD4+ TH cell activation in primary cells from obese individuals, extending the role of this metabolite to a disease associated with low-grade inflammation. Together, these data identify a nutrient-load responsive SCFA-G protein-coupled receptor linked pathway to regulate CD4+ TH cell immune responsiveness.
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Ácidos Grasos no Esterificados , Propionatos , Humanos , Propionatos/farmacología , Leucocitos Mononucleares , Receptores Acoplados a Proteínas G/genética , ObesidadRESUMEN
Adverse social determinants of health (aSDoH) are associated with obesity and related comorbidities like diabetes, cardiovascular disease, and cancer. Obesity is also associated with natural killer cell (NK) dysregulation, suggesting a potential mechanistic link. Therefore, we measured NK phenotypes and function in a cohort of African-American (AA) women from resource-limited neighborhoods. Obesity was associated with reduced NK cytotoxicity and a shift towards a regulatory phenotype. In vitro, LDL promoted NK dysfunction, implicating hyperlipidemia as a mediator of obesity-related immune dysregulation. Dual specific phosphatase 1 (DUSP1) was induced by LDL and was upregulated in NK cells from subjects with obesity, implicating DUSP1 in obesity-mediated NK dysfunction. In vitro, DUSP1 repressed LAMP1/CD107a, depleting NK cells of functional lysosomes to prevent degranulation and cytokine secretion. Together, these data provide novel mechanistic links between aSDoH, obesity, and immune dysregulation that could be leveraged to improve outcomes in marginalized populations.
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Granulocyte colony stimulating factor (G-CSF) is commonly used as adjunct treatment to hasten recovery from neutropenia following chemotherapy and autologous transplantation of hematopoietic stem and progenitor cells (HSPCs) for malignant disorders. However, the utility of G-CSF administration after ex vivo gene therapy procedures targeting human HSPCs has not been thoroughly evaluated. Here, we provide evidence that post-transplant administration of G-CSF impedes engraftment of CRISPR-Cas9 gene edited human HSPCs in xenograft models. G-CSF acts by exacerbating the p53-mediated DNA damage response triggered by Cas9- mediated DNA double-stranded breaks. Transient p53 inhibition in culture attenuates the negative impact of G-CSF on gene edited HSPC function. In contrast, post-transplant administration of G-CSF does not impair the repopulating properties of unmanipulated human HSPCs or HSPCs genetically engineered by transduction with lentiviral vectors. The potential for post-transplant G-CSF administration to aggravate HSPC toxicity associated with CRISPR-Cas9 gene editing should be considered in the design of ex vivo autologous HSPC gene editing clinical trials.
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Persistent skin inflammation and impaired resolution are the main contributors to psoriasis and associated cardiometabolic complications. Omega-3 polyunsaturated fatty acids (PUFAs), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), are known to exert beneficial effects on inflammatory response and lipid function. However, a specific role of omega-3 PUFAs in psoriasis and accompanied pathologies are still a matter of debate. Here, we carried out a direct comparison between EPA and DHA 12 weeks diet intervention treatment of psoriasis-like skin inflammation in the K14-Rac1V12 mouse model. By utilizing sensitive techniques, we targeted EPA- and DHA-derived specialized pro-resolving lipid mediators and identified tightly connected signaling pathways by RNA sequencing. Treatment with experimental diets significantly decreased circulating pro-inflammatory cytokines and bioactive lipid mediators, altered psoriasis macrophage phenotypes and genes of lipid oxidation. The superficial role of these changes was related to DHA treatment and included increased levels of resolvin D5, protectin DX and maresin 2 in the skin. EPA treated mice had less pronounced effects but demonstrated a decreased skin accumulation of prostaglandin E2 and thromboxane B2. These results indicate that modulating psoriasis skin inflammation with the omega-3 PUFAs may have clinical significance and DHA treatment might be considered over EPA in this specific disease.
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Ácidos Grasos Omega-3 , Psoriasis , Ratones , Animales , Ácidos Docosahexaenoicos/farmacología , Ácidos Docosahexaenoicos/metabolismo , Ácidos Grasos Omega-3/farmacología , Ácidos Grasos Omega-3/metabolismo , Ácido Eicosapentaenoico/farmacología , Ácido Eicosapentaenoico/metabolismo , Dieta , Inflamación/metabolismo , Psoriasis/tratamiento farmacológico , Ácidos Grasos/metabolismoRESUMEN
No human vaccine is available for visceral leishmaniasis (VL). Live attenuated centrin gene-deleted L. donovani (LdCen-/-) parasite vaccine has been shown to induce robust innate immunity and provide protection in animal models. Toll-like receptors (TLRs) are expressed in innate immune cells and are essential for the early stages of Leishmania infection. Among TLRs, TLR-9 signaling has been reported to induce host protection during Leishmania infection. Importantly, TLR-9 ligands have been used as immune enhancers for non-live vaccination strategies against leishmaniasis. However, the function of TLR-9 in the generation of a protective immune response in live attenuated Leishmania vaccines remains unknown. In this study, we investigated the function of TLR-9 during LdCen-/- infection and found that it increased the expression of TLR-9 on DCs and macrophages from ear-draining lymph nodes and spleen. The increase in TLR-9 expression resulted in changes in downstream signaling in DCs mediated through signaling protein myeloid differentiation primary response 88 (MyD88), resulting in activation and nuclear translocation of nuclear factor-κB (NF-κB). This process resulted in an increase in the DC's proinflammatory response, activation, and DC-mediated CD4+T cell proliferation. Further, LdCen-/- immunization in TLR-9-/- mice resulted in a significant loss of protective immunity. Thus, LdCen-/- vaccine naturally activates the TLR-9 signaling pathway to elicit protective immunity against virulent L. donovani challenge.
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Hyponatremia and salt wasting is a common occurance in patients with HIV/AIDS, however, the understanding of its contributing factors is limited. HIV viral protein R (Vpr) contributes to HIV-associated nephropathy. To investigate the effects of Vpr on the expression level of the Slc12a3 gene, encoding the Na-Cl cotransporter, which is responsible for sodium reabsorption in distal nephron segments, we performed single-nucleus RNA sequencing of kidney cortices from three wild-type (WT) and three Vpr-transgenic (Vpr Tg) mice. The results showed that the percentage of distal convoluted tubule (DCT) cells was significantly lower in Vpr Tg mice compared with WT mice (P < 0.05), and that in Vpr Tg mice, Slc12a3 expression was not different in DCT cell cluster. The Pvalb+ DCT1 subcluster had fewer cells in Vpr Tg mice compared with WT (P < 0.01). Immunohistochemistry demonstrated fewer Slc12a3+ Pvalb+ DCT1 segments in Vpr Tg mice. Differential gene expression analysis comparing Vpr Tg and WT in the DCT cluster showed Ier3, an inhibitor of apoptosis, to be the most downregulated gene. These observations demonstrate that the salt-wasting effect of Vpr in Vpr Tg mice is mediated by loss of Slc12a3+ Pvalb+ DCT1 segments via apoptosis dysregulation.
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Arginine-specific mono-ADP-ribosylation is a reversible post-translational modification; arginine-specific, cholera toxin-like mono-ADP-ribosyltransferases (ARTCs) transfer ADP-ribose from NAD + to arginine, followed by cleavage of ADP-ribose-(arginine)protein bond by ADP-ribosylarginine hydrolase 1 (ARH1), generating unmodified (arginine)protein. ARTC1 has been shown to enhance tumorigenicity as does Arh1 deficiency. In this study, Artc1 -KO and Artc1/Arh1 -double-KO mice showed decreased spontaneous tumorigenesis and increased age-dependent, multi-organ inflammation with upregulation of pro-inflammatory cytokine TNF- α . In a xenograft model using tumorigenic Arh1 -KO mouse embryonic fibroblasts (MEFs), tumorigenicity was decreased in Artc1 -KO and heterozygous recipient mice, with tumor infiltration by CD8 + T cells and macrophages, leading to necroptosis, suggesting that ARTC1 promotes the tumor microenvironment. Furthermore, Artc1/Arh1 -double-KO MEFs showed decreased tumorigenesis in nude mice, showing that tumor cells as well as tumor microenvironment require ARTC1. By echocardiography and MRI, Artc1 -KO and heterozygous mice showed male-specific, reduced myocardial contractility. Furthermore, Artc1 -KO male hearts exhibited enhanced susceptibility to myocardial ischemia-reperfusion-induced injury with increased receptor-interacting protein kinase 3 (RIP3) protein levels compared to WT mice, suggesting that ARTC1 suppresses necroptosis. Overall survival rate of Artc1 -KO was less than their Artc1 -WT counterparts, primarily due to enhanced immune response and inflammation. Thus, anti-ARTC1 agents may reduce tumorigenesis but may increase multi-organ inflammation and decrease cardiac contractility.
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OBJECTIVE: Colchicine is known to reduce inflammation and improve endothelial cell function and atherosclerosis in obesity, but there is little knowledge of the specific circulating leukocyte populations that are modulated by colchicine. METHODS: A secondary analysis of a double-blind randomized controlled trial of colchicine 0.6 mg or placebo twice daily for 3 months on circulating leukocyte populations and regulation of the immune secretome in 35 adults with obesity was performed. RESULTS: Colchicine altered multiple innate immune cell populations, including dendritic cells and lymphoid progenitor cells, monocytes, and natural killer cells when compared with placebo. Among all subjects and within the colchicine group, changes in natural killer cells were significantly positively associated with reductions in biomarkers of inflammation, including cyclooxygenase 2, pulmonary surfactant-associated protein D, myeloperoxidase, proteinase 3, interleukin-16, and resistin. Changes in dendritic cells were positively correlated with changes in serum heart-type fatty acid-binding protein concentrations. Additionally, colchicine treatment reduced cluster of differentiation (CD) CD4+ T effector cells and CD8+ T cytotoxic cells. Conversely, colchicine increased CD4+ and CD8+ T central memory cells and activated CD38High CD8+ T cells. Changes in CD4+ T effector cells were associated with changes in serum heart-type fatty acid-binding protein. CONCLUSIONS: In adults with obesity, colchicine significantly affects circulating leukocyte populations involved in both innate and adaptive immune systems along with the associated inflammatory secretome.
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Colchicina , Leucocitos Mononucleares , Adulto , Humanos , Colchicina/farmacología , Colchicina/uso terapéutico , Obesidad/complicaciones , Inflamación/metabolismo , Proteínas de Unión a Ácidos Grasos/uso terapéuticoRESUMEN
Small intestinal neuroendocrine tumors (SI-NETs) are serotonin-secreting well-differentiated neuroendocrine tumors of putative enterochromaffin (EC) cell origin. However, EC cell-derived tumorigenesis remains poorly understood. Here, we examined whether the gain of Myc and the loss of RB1 and Trp53 function in EC cells result in SI-NET using tryptophan hydroxylase 1 (TPH1) Cre-ERT2-driven RB1fl Trp53fl MycLSL (RPM) mice. TPH1-Cre-induced gain of Myc and loss of RB1 and Trp53 function resulted in endocrine or neuronal tumors in pancreas, lung, enteric neurons, and brain. Lineage tracing indicated that the cellular origin for these tumors was TPH1-expressing neuroendocrine, neuronal, or their precursor cells in these organs. However, despite that TPH1 is most highly expressed in EC cells of the small intestine, we observed no incidence of EC cell tumors. Instead, the tumor of epithelial cell origin in the intestine was exclusively nonendocrine adenocarcinoma, suggesting dedifferentiation of EC cells into intestinal stem cells (ISCs) as a cellular mechanism. Furthermore, ex vivo organoid studies indicated that loss of functions of Rb1 and Trp53 accelerated dedifferentiation of EC cells that were susceptible to apoptosis with expression of activated MycT58A, suggesting that the rare dedifferentiating cells escaping cell death went on to develop adenocarcinomas. Lineage tracing demonstrated that EC cells in the small intestine were short-lived compared with neuroendocrine or neuronal cells in other organs. In contrast, EC cell-derived ISCs were long-lasting and actively cycling and thus susceptible to transformation. These results suggest that tissue- and cell-specific properties of EC cells such as rapid cell turnover and homeostatic dedifferentiation, affect the fate and rate of tumorigenesis induced by genetic alterations and provide important insights into EC cell-derived tumorigenesis.NEW & NOTEWORTHY Small intestinal neuroendocrine tumors are of putative enterochromaffin (EC) cell origin and are the most common malignancy in the small intestine, followed by adenocarcinoma. However, the tumorigenesis of these tumor types remains poorly understood. The present lineage tracing studies showed that tissue- and cell-specific properties of EC cells such as rapid cell turnover and homeostatic dedifferentiation affect the fate and rate of tumorigenesis induced by genetic alterations toward a rare occurrence of adenocarcinoma.
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Adenocarcinoma , Neoplasias Intestinales , Tumores Neuroendocrinos , Ratones , Animales , Células Enterocromafines/metabolismo , Intestino Delgado/patología , Carcinogénesis/metabolismo , Transformación Celular Neoplásica/metabolismo , Neoplasias Intestinales/metabolismo , Tumores Neuroendocrinos/metabolismo , Adenocarcinoma/genéticaRESUMEN
Sickle cell disease (SCD) and ß-thalassemia are among the most common genetic disorders worldwide, affecting global health and mortality. Hemoglobin A2 (HbA2, α2δ2) is expressed at a low level in adult blood due to the lack of the Kruppel-like factor 1 (KLF1) binding motif in the δ-globin promoter region. However, HbA2 is fully functional as an oxygen transporter, and could be a valid antisickling agent in SCD, as well as a substitute for hemoglobin A in ß-thalassemia. We have previously demonstrated that KLF1-GATA1 fusion protein could interact with the δ-globin promoter and increase δ-globin expression in human primary CD34+ cells. We report the effects of 2 KLF1-GATA1 fusion proteins on hemoglobin expression, as well as SCD phenotypic correction in vitro and in vivo. Forced expression of KLF1-GATA1 fusion protein enhanced δ-globin gene and HbA2 expression, as well as reduced hypoxia-related sickling, in erythroid cells cultured from both human sickle CD34+ cells and SCD mouse hematopoietic stem cells (HSCs). The fusion proteins had no impact on erythroid cell differentiation, proliferation, and enucleation. Transplantation of highly purified SCD mouse HSCs expressing KLF1-GATA1 fusion protein into SCD mice lessened the severity of the anemia, reduced the sickling of red blood cells, improved SCD-related pathological alterations in spleen, kidney, and liver, and restored urine-concentrating ability in recipient mice. Taken together, these results indicate that the use of KLF1-GATA1 fusion constructs may represent a new gene therapy approach for hemoglobinopathies.
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Anemia de Células Falciformes , Proteínas Recombinantes de Fusión , Talasemia beta , Globinas delta , Animales , Humanos , Ratones , Anemia de Células Falciformes/genética , Anemia de Células Falciformes/terapia , Antígenos CD34/metabolismo , Talasemia beta/genética , Globinas delta/genética , Factor de Transcripción GATA1/genética , Fenotipo , Proteínas Recombinantes de Fusión/uso terapéuticoRESUMEN
BACKGROUND: Neutrophils are involved in the initial host responses to pathogens. Neutrophils can activate T cell responses either independently or through indirect involvement of Dendritic cells (DCs). Recently we have demonstrated direct neutrophil-T cell interactions that initiate adaptive immune responses following immunization with live attenuated Leishmania donovani centrin deleted parasite vaccine (LdCen-/-). However, neutrophil-DC interactions in T cell priming in vaccine immunity in general are not known. In this study we evaluated the interaction between neutrophils and DCs during LdCen-/- infection and compared with wild type parasite (LdWT) both in vitro and in vivo. METHODOLOGY/FINDINGS: LdCen-/- parasite induced increased expression of CCL3 in neutrophils caused higher recruitment of DCs capable of inducing a strong proinflammatory response and elevated co-stimulatory molecule expression compared to LdWT infection. To further illustrate neutrophil-DCs interactions in vivo, we infected LYS-eGFP mice with red fluorescent LdWT/LdCen-/- parasites and sort selected DCs that engulfed the neutrophil containing parasites or DCs that acquired the parasites directly in the ear draining lymph nodes (dLN) 5d post infection. The DCs predominantly acquired the parasites by phagocytosing infected neutrophils. Specifically, DCs containing LdCen-/- parasitized neutrophils exhibited a proinflammatory phenotype, increased expression of costimulatory molecules and initiated higher CD4+T cell priming ex-vivo. Notably, potent DC activation occurred when LdCen-/- parasites were acquired indirectly via engulfment of parasitized neutrophils compared to direct engulfment of LdCen-/- parasites by DCs. Neutrophil depletion in LdCen-/- infected mice significantly abrogated expression of CCL3 resulting in decreased DC recruitment in ear dLN. This event led to poor CD4+Th1 cell priming ex vivo that correlated with attenuated Tbet expression in ear dLN derived CD4+ T cells in vivo. CONCLUSIONS: Collectively, LdCen-/- containing neutrophils phagocytized by DC markedly influence the phenotype and antigen presenting capacity of DCs early on and thus play an immune-regulatory role in shaping vaccine induced host protective response.
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Leishmania donovani , Vacunas contra la Leishmaniasis , Leishmaniasis Visceral , Animales , Comunicación Celular , Células Dendríticas , Leishmania donovani/fisiología , Leishmaniasis Visceral/parasitología , Ratones , Neutrófilos , Vacunas AtenuadasRESUMEN
OBJECTIVE: The aim of this study was to examine whether colchicine's anti-inflammatory effects would improve measures of lipolysis and distribution of leukocyte populations in subcutaneous adipose tissue (SAT). METHODS: A secondary analysis was conducted for a double-blind, randomized, placebo-controlled pilot study in which 40 adults with obesity and metabolic syndrome (MetS) were randomized to colchicine 0.6 mg or placebo twice daily for 3 months. Non-insulin-suppressible (l0 ), insulin-suppressible (l2 ), and maximal (l0 +l2 ) lipolysis rates were calculated by minimal model analysis. Body composition was determined by dual-energy x-ray absorptiometry. SAT leukocyte populations were characterized by flow cytometry analysis from biopsied samples obtained before and after the intervention. RESULTS: Colchicine treatment significantly decreased l2 and l0 +l2 versus placebo (p < 0.05). These changes were associated with a significant reduction in markers of systemic inflammation, including high-sensitivity C-reactive protein, resistin, and circulating monocytes and neutrophils (p < 0.01). Colchicine did not significantly alter SAT leukocyte population distributions (p > 0.05). CONCLUSIONS: In adults with obesity and MetS, colchicine appears to improve insulin regulation of lipolysis and reduce markers of systemic inflammation independent of an effect on local leukocyte distributions in SAT. Further studies are needed to better understand the mechanisms by which colchicine affects adipose tissue metabolic pathways in adults with obesity and MetS.
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Resistencia a la Insulina , Síndrome Metabólico , Tejido Adiposo/metabolismo , Adulto , Biomarcadores/metabolismo , Colchicina/metabolismo , Colchicina/farmacología , Colchicina/uso terapéutico , Humanos , Inflamación/metabolismo , Insulina/metabolismo , Lipólisis , Síndrome Metabólico/metabolismo , Obesidad/complicaciones , Obesidad/tratamiento farmacológico , Obesidad/metabolismoRESUMEN
BACKGROUND: The powerful 'graft versus leukemia' effect thought partly responsible for the therapeutic effect of allogeneic hematopoietic cell transplantation in acute myeloid leukemia (AML) provides rationale for investigation of immune-based therapies in this high-risk blood cancer. There is considerable preclinical evidence for potential synergy between PD-1 immune checkpoint blockade and the hypomethylating agents already commonly used for this disease. METHODS: We report here the results of 17 H-0026 (PD-AML, NCT02996474), an investigator sponsored, single-institution, single-arm open-label 10-subject pilot study to test the feasibility of the first-in-human combination of pembrolizumab and decitabine in adult patients with refractory or relapsed AML (R-AML). RESULTS: In this cohort of previously treated patients, this novel combination of anti-PD-1 and hypomethylating therapy was feasible and associated with a best response of stable disease or better in 6 of 10 patients. Considerable immunological changes were identified using T cell receptor ß sequencing as well as single-cell immunophenotypic and RNA expression analyses on sorted CD3+ T cells in patients who developed immune-related adverse events (irAEs) during treatment. Clonal T cell expansions occurred at irAE onset; single-cell sequencing demonstrated that these expanded clones were predominately CD8+ effector memory T cells with high cell surface PD-1 expression and transcriptional profiles indicative of activation and cytotoxicity. In contrast, no such distinctive immune changes were detectable in those experiencing a measurable antileukemic response during treatment. CONCLUSION: Addition of pembrolizumab to 10-day decitabine therapy was clinically feasible in patients with R-AML, with immunological changes from PD-1 blockade observed in patients experiencing irAEs.
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Anticuerpos Monoclonales Humanizados/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Decitabina/uso terapéutico , Inmunoterapia/métodos , Leucemia Mieloide Aguda/tratamiento farmacológico , Anticuerpos Monoclonales Humanizados/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Estudios de Cohortes , Decitabina/farmacología , Femenino , Humanos , Masculino , Proyectos Piloto , RecurrenciaRESUMEN
Haploidentical hematopoietic stem cell transplantation (haplo-HSCT) is a widely available curative option for patients with sickle cell disease (SCD). Our original non-myeloablative haplo-HSCT trial employing post-transplant (PT) cyclophosphamide had a low incidence of GVHD but had high rejection rates. Here, we aimed to evaluate immune reconstitution following haplo-HSCT and identify cytokines and cells associated with graft rejection/engraftment. 50 cytokines and 10 immune cell subsets were screened using multiplex-ELISA and flow cytometry, respectively, at baseline and PT-Days 30, 60, 100, and 180. We observed the most significant differences in cytokine levels between the engrafted and rejected groups at PT-Day 60, corresponding with clinical findings of secondary graft rejection. Of the 44 cytokines evaluated, plasma concentrations of 19 cytokines were different between the two groups at PT-Day 60. Factor analysis suggested two independent factors. The first factor (IL-17A, IL-10, IL-7, G-CSF, IL-2, MIP-1a, VEGF, and TGFb1 contributed significantly) was strongly associated with engraftment with OR = 2.7 (95%CI of 1.4 to 5.4), whereas the second factor (GROa and IL-18 contributed significantly) was not significantly associated with engraftment. Sufficient donor myeloid chimerism (DMC) is critical for the success of HSCT; here, we evaluated immune cells among high (H) DMC (DMC≥20%) and low (L) DMC (DMC<20%) groups along with engrafted and rejected groups. We found that early myeloid-derived suppressor cell (eMDSC) frequencies were elevated in engrafted patients and patients with HDMC at PT-Day 30 (P< 0.04 & P< 0.003, respectively). 9 of 20 patients were evaluated for the source of eMDSCs. The HDMC group had high mixed chimeric eMDSCs as compared to the LDMC group (P< 0.00001). We found a positive correlation between the frequencies of eMDSCs and Tregs at PT-Day 100 (r=0.72, P <0.0007); eMDSCs at BSL and Tregs at PT-Day 100 (r=0.63, P <0.004). Of 10 immune regulatory cells and 50 cytokines, we observed mixed chimeric eMDSCs and IL-17A, IL-10, IL-7, G-CSF, IL-2, MIP-1a, VEGF, TGFb1 as potential hits which could serve as prognostic markers in predicting allograft outcome towards engraftment following haploidentical HSCT employing post-transplant cyclophosphamide. The current findings need to be replicated and further explored in a larger cohort.
Asunto(s)
Anemia de Células Falciformes/terapia , Trasplante de Células Madre Hematopoyéticas , Reconstitución Inmune/inmunología , Quimera por Trasplante , Adulto , Anemia de Células Falciformes/inmunología , Quimerismo , Ciclofosfamida/uso terapéutico , Citocinas/inmunología , Rechazo de Injerto/inmunología , Supervivencia de Injerto/inmunología , Humanos , Inmunosupresores/uso terapéutico , Células Supresoras de Origen Mieloide , Pronóstico , Acondicionamiento Pretrasplante , Trasplante Haploidéntico , Resultado del TratamientoRESUMEN
Genetic mutations associated with acute myeloid leukemia (AML) also occur in age-related clonal hematopoiesis, often in the same individual. This makes confident assignment of detected variants to malignancy challenging. The issue is particularly crucial for AML post-treatment measurable residual disease monitoring, where results can be discordant between genetic sequencing and flow cytometry. We show here, that it is possible to distinguish AML from clonal hematopoiesis and to resolve the immunophenotypic identity of clonal architecture. To achieve this, we first design patient-specific DNA probes based on patient's whole-genome sequencing, and then use them for patient-personalized single-cell DNA sequencing with simultaneous single-cell antibody-oligonucleotide sequencing. Examples illustrate AML arising from DNMT3A and TET2 mutated clones as well as independently. The ability to personalize single-cell proteogenomic assessment for individual patients based on leukemia-specific genomic features has implications for ongoing AML precision medicine efforts.