RESUMEN
Substance use disorders (SUD) and drug addiction are major threats to public health, impacting not only the millions of individuals struggling with SUD, but also surrounding families and communities. One of the seminal challenges in treating and studying addiction in human populations is the high prevalence of co-morbid conditions, including an increased risk of contracting a human immunodeficiency virus (HIV) infection. Of the ~15 million people who inject drugs globally, 17% are persons with HIV. Conversely, HIV is a risk factor for SUD because chronic pain syndromes, often encountered in persons with HIV, can lead to an increased use of opioid pain medications that in turn can increase the risk for opioid addiction. We hypothesize that SUD and HIV exert shared effects on brain cell types, including adaptations related to neuroplasticity, neurodegeneration, and neuroinflammation. Basic research is needed to refine our understanding of these affected cell types and adaptations. Studying the effects of SUD in the context of HIV at the single-cell level represents a compelling strategy to understand the reciprocal interactions among both conditions, made feasible by the availability of large, extensively-phenotyped human brain tissue collections that have been amassed by the Neuro-HIV research community. In addition, sophisticated animal models that have been developed for both conditions provide a means to precisely evaluate specific exposures and stages of disease. We propose that single-cell genomics is a uniquely powerful technology to characterize the effects of SUD and HIV in the brain, integrating data from human cohorts and animal models. We have formed the Single-Cell Opioid Responses in the Context of HIV (SCORCH) consortium to carry out this strategy.
RESUMEN
CXCR4 is a key regulator of the development of NK cells and DCs, both of which play an important role in early placental development and immune tolerance at the maternal-fetal interface. However, the role of CXCR4 in pregnancy is not well understood. Our study demonstrates that adult-induced global genetic CXCR4 deletion, but not uterine-specific CXCR4 deletion, was associated with increased pregnancy resorptions and decreased litter size. CXCR4-deficient mice had decreased NK cells and increased granulocytes in the decidua, along with increased leukocyte numbers in peripheral blood. We found that CXCR4-deficient mice had abnormal decidual NK cell aggregates and NK cell infiltration into trophoblast areas beyond the giant cell layer. This was associated with low NK cell expression of granzyme B, a NK cell granule effector, indicative of NK cell dysfunction. Pregnancy failure in these mice was associated with abnormalities in placental vascular development and increased placental expression of inflammatory genes. Importantly, adoptive BM transfer of WT CXCR4+ BM cells into CXCR4-deficient mice rescued the reproductive deficits by normalizing NK cell function and mediating normal placental vascular development. Collectively, our study found an important role for maternal CXCR4 expression in immune cell function, placental development, and pregnancy maintenance.
Asunto(s)
Decidua , Placenta , Animales , Femenino , Ratones , Embarazo , Placentación/genética , Transducción de Señal/fisiología , Trofoblastos/metabolismoRESUMEN
Fibrosis is regulated by interactions between immune and mesenchymal cells. However, the capacity of cell types to modulate human fibrosis pathology is poorly understood due to lack of a fully humanized model system. MISTRG6 mice were engineered by homologous mouse/human gene replacement to develop an immune system like humans when engrafted with human hematopoietic stem cells (HSCs). We utilized MISTRG6 mice to model scleroderma by transplantation of healthy or scleroderma skin from a patient with pansclerotic morphea to humanized mice engrafted with unmatched allogeneic HSC. We identified that scleroderma skin grafts contained both skin and bone marrow-derived human CD4 and CD8 T cells along with human endothelial cells and pericytes. Unlike healthy skin, fibroblasts in scleroderma skin were depleted and replaced by mouse fibroblasts. Furthermore, HSC engraftment alleviated multiple signatures of fibrosis, including expression of collagen and interferon genes, and proliferation and activation of human T cells. Fibrosis improvement correlated with reduced markers of T cell activation and expression of human IL-6 by mesenchymal cells. Mechanistic studies supported a model whereby IL-6 trans-signaling driven by CD4 T cell-derived soluble IL-6 receptor complexed with fibroblast-derived IL-6 promoted excess extracellular matrix gene expression. Thus, MISTRG6 mice transplanted with scleroderma skin demonstrated multiple fibrotic responses centered around human IL-6 signaling, which was improved by the presence of healthy bone marrow-derived immune cells. Our results highlight the importance of IL-6 trans-signaling in pathogenesis of scleroderma and the ability of healthy bone marrow-derived immune cells to mitigate disease.
Asunto(s)
Basidiomycota , Esclerodermia Localizada , Humanos , Animales , Ratones , Interleucina-6 , Células Endoteliales , Piel , Modelos Animales de EnfermedadRESUMEN
The neonatal Fc receptor (FcRn) is responsible for recycling of IgG antibodies and albumin throughout the body. This mechanism has been exploited for pharmaceutic delivery across an array of diseases to either enhance or diminish this function. Monoclonal antibodies and albumin-bound nanoparticles are examples of FcRn-dependent anti-cancer therapeutics. Despite its importance in drug delivery, little is known about FcRn expression in circulating immune cells. Through time-of-flight mass cytometry (CyTOF) we were able to characterize FcRn expression in peripheral blood mononuclear cell (PBMC) populations of pancreatic ductal adenocarcinoma (PDAC) patients and non-cancer donors. Furthermore, we were able to replicate these findings in an orthotopic murine model of PDAC. Altogether, we found that in both patients and mice with PDAC, FcRn was elevated in migratory and resident classical dendritic cell type 2 (cDC2) as well as monocytic and granulocytic myeloid-derived suppressor cell (MDSC) populations compared to tumor-free controls. Furthermore, PBMCs from PDAC patients had elevated monocyte, dendritic cells and MDSCs relative to non-cancer donor PBMCs. Future investigations into FcRn activity may further elucidate possible mechanisms of poor efficacy of antibody immunotherapies in patients with PDAC.
Asunto(s)
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Albúminas , Animales , Antígenos de Histocompatibilidad Clase I , Leucocitos Mononucleares/metabolismo , Ratones , Monocitos/metabolismo , Receptores Fc , Neoplasias PancreáticasRESUMEN
BACKGROUND: Respiratory failure (RF) is a common cause of death and morbid complication in trauma patients. Extracorporeal membrane oxygenation (ECMO) is increasingly used in adults with RF refractory to invasive mechanical ventilation. However, use of ECMO remains limited for this patient population as they often have contraindications for anticoagulation. STUDY DESIGN: Medical records were retroactively searched for all adult patients who were admitted to the trauma service and received veno-venous ECMO (VV ECMO) support between June 2015 and August 2018. Survival to discharge and ECMO-related complications were collected and analyzed. RESULTS: Fifteen patients from a large Level I trauma center met the criteria. The median PaO2/FiO2 ratio was 53.0 (IQR, 27.0-76.0), median injury severity score was 34.0 (IQR, 27.0-43.0), and the median duration of ECMO support was 11 days (IQR, 7.5-20.0). For this cohort, the survival-to-discharge rate was 87% (13/15). The incidence of neurologic complications was 13%, and deep vein thrombosis was reported in two cases (13%). CONCLUSIONS: Survival rates of trauma patients in this study are equivalent to, or may exceed, those of non-trauma patients who receive ECMO support for other types of RF. With the employment of a multidisciplinary team assessment and proper patient selection, early cannulation, traumatic RF may be safely supported with VV ECMO in experienced centers.
Asunto(s)
Oxigenación por Membrana Extracorpórea , Insuficiencia Respiratoria , Adulto , Oxigenación por Membrana Extracorpórea/efectos adversos , Humanos , Alta del Paciente , Respiración Artificial , Insuficiencia Respiratoria/etiología , Insuficiencia Respiratoria/terapia , Estudios Retrospectivos , Resultado del TratamientoRESUMEN
Glioblastoma multiforme (GBM) is the most lethal primary brain tumor characterized by high cellular and molecular heterogeneity, hypervascularization, and innate drug resistance. Cellular components and extracellular matrix (ECM) are the two primary sources of heterogeneity in GBM. Here, biomimetic tri-regional GBM models with tumor regions, acellular ECM regions, and an endothelial region with regional stiffnesses patterned corresponding to the GBM stroma, pathological or normal brain parenchyma, and brain capillaries, are developed. Patient-derived GBM cells, human endothelial cells, and hyaluronic acid derivatives are used to generate a species-matched and biochemically relevant microenvironment. This in vitro study demonstrates that biophysical cues are involved in various tumor cell behaviors and angiogenic potentials and promote different molecular subtypes of GBM. The stiff models are enriched in the mesenchymal subtype, exhibit diffuse invasion of tumor cells, and induce protruding angiogenesis and higher drug resistance to temozolomide. Meanwhile, the soft models demonstrate enrichment in the classical subtype and support expansive cell growth. The three-dimensional bioprinting technology utilized in this study enables rapid, flexible, and reproducible patient-specific GBM modeling with biophysical heterogeneity that can be employed by future studies as a tunable system to interrogate GBM disease mechanisms and screen drug compounds.
Asunto(s)
Bioimpresión , Neoplasias Encefálicas , Glioblastoma , Línea Celular Tumoral , Células Endoteliales , Humanos , Microambiente TumoralRESUMEN
Methamphetamine (METH) is a potent stimulant that induces a euphoric state but also causes cognitive impairment, neurotoxicity and neurodevelopmental deficits. Yet, the molecular mechanisms by which METH causes neurodevelopmental defects have remained elusive. Here we utilized human cerebral organoids and single-cell RNA sequencing (scRNA-seq) to study the effects of prenatal METH exposure on fetal brain development. We analyzed 20,758 cells from eight untreated and six METH-treated cerebral organoids and found that the organoids developed from embryonic stem cells contained a diverse array of glial and neuronal cell types. We further identified transcriptionally distinct populations of astrocytes and oligodendrocytes within cerebral organoids. Treatment of organoids with METH-induced marked changes in transcription in multiple cell types, including astrocytes and neural progenitor cells. METH also elicited novel astrocyte-specific gene expression networks regulating responses to cytokines, and inflammasome. Moreover, upregulation of immediate early genes, complement factors, apoptosis, and immune response genes suggests a neuroinflammatory program induced by METH regulating neural stem cell proliferation, differentiation, and cell death. Finally, we observed marked METH-induced changes in neuroinflammatory and cytokine gene expression at the RNA and protein levels. Our data suggest that human cerebral organoids represent a model system to study drug-induced neuroinflammation at single-cell resolution.
Asunto(s)
Estimulantes del Sistema Nervioso Central , Metanfetamina , Astrocitos , Humanos , Neuronas , OrganoidesRESUMEN
An impressive clinical success has been observed in treating a variety of cancers using immunotherapy with programmed cell death-1 (PD-1) checkpoint blockade. However, limited response in most patients treated with anti-PD-1 antibodies remains a challenge, requiring better understanding of molecular mechanisms limiting immunotherapy. In colorectal cancer (CRC) resistant to immunotherapy, mismatch-repair-proficient or microsatellite instability-low (pMMR-MSI-L) tumors have low mutation burden and constitute ~85% of patients. Here, we show that inhibition of N6 -methyladenosine (m6 A) mRNA modification by depletion of methyltransferases, Mettl3 and Mettl14, enhanced response to anti-PD-1 treatment in pMMR-MSI-L CRC and melanoma. Mettl3- or Mettl14-deficient tumors increased cytotoxic tumor-infiltrating CD8+ T cells and elevated secretion of IFN-γ, Cxcl9, and Cxcl10 in tumor microenvironment in vivo. Mechanistically, Mettl3 or Mettl14 loss promoted IFN-γ-Stat1-Irf1 signaling through stabilizing the Stat1 and Irf1 mRNA via Ythdf2. Finally, we found a negative correlation between METTL3 or METTL14 and STAT1 in 59 patients with pMMR-MSI-L CRC tumors. Altogether, our findings uncover a new awareness of the function of RNA methylation in adaptive immunity and provide METTL3 and METTL14 as potential therapeutic targets in anticancer immunotherapy.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Neoplasias Colorrectales/enzimología , Citocinas/metabolismo , Inmunoterapia/métodos , Melanoma/enzimología , Metiltransferasas/metabolismo , Receptor de Muerte Celular Programada 1/metabolismo , Adenosina/análogos & derivados , Adenosina/metabolismo , Animales , Linfocitos T CD8-positivos/inmunología , Línea Celular Tumoral , Proliferación Celular/genética , Quimiocina CXCL10 , Quimiocina CXCL9/metabolismo , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/inmunología , Neoplasias Colorrectales/metabolismo , Humanos , Inhibidores de Puntos de Control Inmunológico , Inmunohistoquímica , Factor 1 Regulador del Interferón/genética , Factor 1 Regulador del Interferón/metabolismo , Interferón gamma/metabolismo , Melanoma/genética , Melanoma/inmunología , Melanoma/metabolismo , Metiltransferasas/genética , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , RNA-Seq , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT1/metabolismo , Microambiente Tumoral/genéticaRESUMEN
Chronic infection with human immunodeficiency virus (HIV) can cause progressive loss of immune cell function, or exhaustion, which impairs control of virus replication. However, little is known about the development and maintenance, as well as heterogeneity of immune cell exhaustion. Here, we investigated the effects of HIV infection on immune cell exhaustion at the transcriptomic level by analyzing single-cell RNA sequencing of peripheral blood mononuclear cells from four healthy subjects (37,847 cells) and six HIV-infected donors (28,610 cells). We identified nine immune cell clusters and eight T cell subclusters, and three of these (exhausted CD4+ and CD8+ T cells and interferon-responsive CD8+ T cells) were detected only in samples from HIV-infected donors. An inhibitory receptor KLRG1 was identified in a HIV-1 specific exhausted CD8+ T cell population expressing KLRG1, TIGIT, and T-betdimEomeshi markers. Ex-vivo antibody blockade of KLRG1 restored the function of HIV-specific exhausted CD8+ T cells demonstrating the contribution of KLRG1+ population to T cell exhaustion and providing an immunotherapy target to treat HIV chronic infection. These data provide a comprehensive analysis of gene signatures associated with immune cell exhaustion during HIV infection, which could be useful in understanding exhaustion mechanisms and developing new cure therapies.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Infecciones por VIH/inmunología , VIH-1/inmunología , Lectinas Tipo C/genética , Receptores Inmunológicos/genética , Análisis de la Célula Individual/métodos , Adulto , Anciano , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/metabolismo , Estudios de Casos y Controles , Femenino , Regulación de la Expresión Génica , Infecciones por VIH/genética , Humanos , Leucocitos Mononucleares/inmunología , Masculino , Persona de Mediana Edad , Análisis de Secuencia de ARN , Adulto JovenRESUMEN
Although immune checkpoint blockade (ICB) therapy has revolutionized cancer treatment, many patients do not respond or develop resistance to ICB. N6 -methylation of adenosine (m6A) in RNA regulates many pathophysiological processes. Here, we show that deletion of the m6A demethylase Alkbh5 sensitized tumors to cancer immunotherapy. Alkbh5 has effects on m6A density and splicing events in tumors during ICB. Alkbh5 modulates Mct4/Slc16a3 expression and lactate content of the tumor microenvironment and the composition of tumor-infiltrating Treg and myeloid-derived suppressor cells. Importantly, a small-molecule Alkbh5 inhibitor enhanced the efficacy of cancer immunotherapy. Notably, the ALKBH5 gene mutation and expression status of melanoma patients correlate with their response to immunotherapy. Our results suggest that m6A demethylases in tumor cells contribute to the efficacy of immunotherapy and identify ALKBH5 as a potential therapeutic target to enhance immunotherapy outcome in melanoma, colorectal, and potentially other cancers.
Asunto(s)
Desmetilasa de ARN, Homólogo 5 de AlkB/metabolismo , Vacunas contra el Cáncer/inmunología , Lactatos/metabolismo , Melanoma/metabolismo , Receptor de Muerte Celular Programada 1/inmunología , Linfocitos T Reguladores/fisiología , Desmetilasa de ARN, Homólogo 5 de AlkB/genética , Anticuerpos , Citocinas/genética , Citocinas/metabolismo , Eliminación de Gen , Regulación Neoplásica de la Expresión Génica , Humanos , Melanoma/terapia , Metiltransferasas/genética , Metiltransferasas/metabolismo , Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Células Supresoras de Origen Mieloide/fisiología , Sitios de Empalme de ARN , Empalme del ARN , Simportadores/genética , Simportadores/metabolismo , Transcriptoma , Microambiente Tumoral , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
BACKGROUND: A significant challenge to overcome in pancreatic ductal adenocarcinoma (PDAC) is the profound systemic immunosuppression that renders this disease non-responsive to immunotherapy. Our supporting data provide evidence that CD200, a regulator of myeloid cell activity, is expressed in the PDAC microenvironment. Additionally, myeloid-derived suppressor cells (MDSC) isolated from patients with PDAC express elevated levels of the CD200 receptor (CD200R). Thus, we hypothesize that CD200 expression in the PDAC microenvironment limits responses to immunotherapy by promoting expansion and activity of MDSC. METHODS: Immunofluorescent staining was used to determine expression of CD200 in murine and human PDAC tissue. Flow cytometry was utilized to test for CD200R expression by immune populations in patient blood samples. In vivo antibody blocking of CD200 was conducted in subcutaneous MT-5 tumor-bearing mice and in a genetically engineered PDAC model (KPC-Brca2 mice). Peripheral blood mononuclear cells (PBMC) from patients with PDAC were analyzed by single-cell RNA sequencing. MDSC expansion assays were completed using healthy donor PBMC stimulated with IL-6/GM-CSF in the presence of recombinant CD200 protein. RESULTS: We found expression of CD200 by human pancreatic cell lines (BxPC3, MiaPaca2, and PANC-1) as well as on primary epithelial pancreatic tumor cells and smooth muscle actin+ stromal cells. CD200R expression was found to be elevated on CD11b+CD33+HLA-DRlo/- MDSC immune populations from patients with PDAC (p=0.0106). Higher expression levels of CD200R were observed in CD15+ MDSC compared with CD14+ MDSC (p<0.001). In vivo studies demonstrated that CD200 antibody blockade limited tumor progression in MT-5 subcutaneous tumor-bearing and in KPC-Brca2 mice (p<0.05). The percentage of intratumoral MDSC was significantly reduced in anti-CD200 treated mice compared with controls. Additionally, in vivo blockade of CD200 can also significantly enhance the efficacy of PD-1 checkpoint antibodies compared with single antibody therapies (p<0.05). Single-cell RNA sequencing of PBMC from patients revealed that CD200R+ MDSC expressed genes involved in cytokine signaling and MDSC expansion. Further, in vitro cytokine-driven expansion and the suppressive activity of human MDSC was enhanced when cocultured with recombinant CD200 protein. CONCLUSIONS: These results indicate that CD200 expression in the PDAC microenvironment may regulate MDSC expansion and that targeting CD200 may enhance activity of checkpoint immunotherapy.
Asunto(s)
Antígenos CD/metabolismo , Carcinoma Ductal Pancreático/inmunología , Terapia de Inmunosupresión , Leucocitos Mononucleares/inmunología , Células Supresoras de Origen Mieloide/inmunología , Neoplasias Pancreáticas/inmunología , Microambiente Tumoral/inmunología , Animales , Antígenos CD/genética , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patología , Modelos Animales de Enfermedad , Humanos , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/patología , Ratones , Células Supresoras de Origen Mieloide/metabolismo , Células Supresoras de Origen Mieloide/patología , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Neoplasias PancreáticasRESUMEN
OBJECTIVES: Patients with cirrhosis have a high rate of 30-day hospital readmission that affects their quality of life and contributes to increased healthcare-related costs. The aim of our study was to identify frequency, predictors, and preventable causes of hospital readmissions among patients with decompensated cirrhosis. METHODS: We retrospectively reviewed electronic medical records of all patients with a confirmed diagnosis of decompensated cirrhosis admitted to Dayton VA Medical Center between 2009 and 2013. Demographics, clinical factors, laboratory values, and outcomes were recorded. Univariate analysis was performed using independent samples t tests and Wilcoxon rank sums tests for continuous variables and χ(2) or Fisher exact tests for categorical variables. A multiple logistic regression analysis was performed for variables found to be significant by univariate analysis to predict the risk factors for 30-day readmission. A detailed chart review was conducted for all patients readmitted within 30 days by a single gastroenterologist to identify the reason for readmission and to decide whether any of these readmissions were preventable. RESULTS: The 30-day readmission rate for decompensated cirrhotic patients was 27.03%. The risk factors for 30-day readmission were higher body mass index (BMI), lower body temperature, higher blood urea nitrogen, higher creatinine, more cirrhosis-related complications, and more readmissions per year per univariate analysis. Multivariable analysis revealed only BMI as a significant predictor of 30-day readmission (P = 0.023). A total of 36.7% of 30-day readmissions were possibly preventable. CONCLUSIONS: The independent variable that predicted 30-day readmission in patients with decompensated cirrhosis was higher BMI. Approximately one-third of 30-day readmissions were possibly preventable. These findings support the need to develop specific interventions for disease management to improve patient care and save on extraneous healthcare costs.