RESUMEN
Aneuploidy, a condition associated with altered chromosome number, hence DNA index, is frequently seen in many diseases including cancers and affects immunity. Iron, an essential nutrient for humans, modulates the immune function and the proliferation of normal and cancer cells. To determine whether impaired immunity seen in iron-deficient subjects may be related to aneuploidy, we measured spleen cell DNA index, percent of cells in different phases of the cell cycle, plasma and/or supernatant IL-2, IL-10, IL-12, and interferon-gamma in control, pair-fed, iron-deficient, and iron-replete mice (N=20-22/group). The test and control diets differed only in iron content (0.09mmol/kg versus 0.9mmol/kg) and were fed for 68days. Mean levels of hemoglobin and liver iron stores of iron-deficient and iron-replete mice were 40-60% lower than those of control and pair-fed mice (P<0.05). Mean plasma levels of IL-10, interferon-gamma and percent of cells in S+G2/M phases were lower in mice with than in those without aneuploidy (P<0.05). Lowest plasma IL-12 and interferon-gamma concentrations were observed in iron-deficient mice with aneuploidy. Mean percents of cultures with aneuploidy and DNA indexes were higher in iron-deficient and iron-replete than in control and pair-fed mice likely due to delayed cell division (P<0.05). Aneuploidy decreased the concentration of IL-2 and interferon-gamma in baseline cultures while it increased that of interferon-gamma in anti-CD3 treated cultures. Aneuploidic indexes negatively correlated with cytokine levels, percents of cells in S+G2/M phases and indicators of iron status (P<0.05). Although chromosome cytogenetics was not performed, for the first time, we report that increased aneuploidy rate may modulate the immune function during iron-deficiency.
Asunto(s)
Aneuploidia , Ciclo Celular , Citocinas/sangre , ADN/metabolismo , Hierro/metabolismo , Animales , Peso Corporal/efectos de los fármacos , Recuento de Células , Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Dieta , Femenino , Linfocitos/citología , Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Mitógenos/farmacología , Modelos Animales , Bazo/citología , Bazo/efectos de los fármacos , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismoRESUMEN
Enhancement of stem cell Ag-1 (Sca-1) expression by myeloid precursors promotes the granulopoietic response to bacterial infection. However, the underlying mechanisms remain unclear. ERK pathway activation strongly enhances proliferation of hematopoietic progenitor cells. In this study, we investigated the role of Sca-1 in promoting ERK-dependent myeloid lineage proliferation and the effects of alcohol on this process. Thirty minutes after i.p. injection of alcohol, mice received i.v. challenge with 5 × 10(7) Escherichia coli for 8 or 24 h. A subset of mice received i.v. BrdU injection 20 h after challenge. Bacteremia increased Sca-1 expression, ERK activation, and proliferation of myeloid and granulopoietic precursors. Alcohol administration suppressed this response and impaired granulocyte production. Sca-1 expression positively correlated with ERK activation and cell cycling, but negatively correlated with myeloperoxidase content in granulopoietic precursors. Alcohol intoxication suppressed ERK activation in granulopoietic precursors and proliferation of these cells during bacteremia. Granulopoietic precursors in Sca-1(-/-) mice failed to activate ERK signaling and could not increase granulomacrophagic CFU activity following bacteremia. These data indicate that Sca-1 expression promotes ERK-dependent myeloid cell proliferation during bacteremia. Suppression of this response could represent an underlying mechanism for developing myelosuppression in alcohol-abusing hosts with severe bacterial infection.
Asunto(s)
Intoxicación Alcohólica/inmunología , Antígenos Ly/inmunología , Bacteriemia/inmunología , Etanol/farmacología , Sistema de Señalización de MAP Quinasas , Proteínas de la Membrana/inmunología , Células Mieloides/metabolismo , Animales , Antígenos Ly/biosíntesis , Antígenos Ly/metabolismo , Proliferación Celular/efectos de los fármacos , Escherichia coli/inmunología , Escherichia coli/patogenicidad , Infecciones por Escherichia coli/inmunología , Granulocitos/efectos de los fármacos , Células Madre Hematopoyéticas/inmunología , Masculino , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Células Mieloides/efectos de los fármacos , Peroxidasa/biosíntesis , Transducción de SeñalRESUMEN
Flow cytometry has the potential to facilitate understanding of the heterogeneous responses of diverse brain cell populations to a variety of stimuli. However, existing methods of applying flow cytometry to brain tissues are each limited in certain ways. They either require genetically labeled cells to achieve separation of specific populations, are not applicable to previously fixed tissue, or are not compatible with downstream mRNA analysis. Here, we describe a group of related methods that overcome many previous limitations and allow robust sorting and downstream molecular analysis of highly enriched populations of specific neuronal and non-neuronal cells from any mammalian brain. We illustrate these techniques, which are compatible with antibodies for both nuclear and non-nuclear epitopes and do not require transgenic animals, with three examples. First, we describe the separation and downstream mRNA analysis of four types of cortical interneurons (somatostatin, parvalbumin, calretinin, and calbindin) from paraformaldehyde-fixed rat brain sections. Second, we demonstrate separation of neurons and non-neurons from zinc-fixed mouse brain cortical sections followed by analysis of enzymatic activity (ACE2 activity) and mRNA expression. Third, we show that routinely fixed post-mortem human brain can be analyzed by isolating parvalbumin-containing neurons from cortical samples that were fixed for periods of up to 8 weeks in formalin. In each case, sorted cell identity was confirmed with mRNA analysis. The neurocytometry methodology described here has the potential to significantly expand studies to analyze the effects of drugs, environmental manipulations, and disease states on the nucleic acid and protein content of specific brain cell populations.
Asunto(s)
Encéfalo/citología , Citometría de Flujo , Neuronas/metabolismo , Enzima Convertidora de Angiotensina 2 , Inhibidores de la Enzima Convertidora de Angiotensina , Animales , Calbindina 2/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas del Tejido Nervioso/metabolismo , Péptidos , Peptidil-Dipeptidasa A/genética , Peptidil-Dipeptidasa A/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Sinapsinas/genética , Sinapsinas/metabolismoRESUMEN
Activated CD8+ T-cells correlate with viral load and may foretell antiretroviral therapy (ART) failure. HIV infection has been suggested to accelerate immunosenescence through chronic persistent inflammation. Alcohol-use disorders (AUD) are prevalent in persons living with HIV/AIDS (PLWHA). We tested the hypothesis that hazardous alcohol consumption accelerates immune activation and immunosenescence. Immune activation and immunosenescence were examined in CD8+ T lymphocytes (CD3+CD4-CD8+) isolated from intestinal biopsies, axillary lymph nodes, and peripheral blood mononuclear cells (PBMCs) of chronic binge alcohol (CBA)-consuming simian immunodeficiency virus (SIV)-infected male rhesus macaques with and without antiretroviral therapy (ART; CBA/ART+, CBA/ART-) and in PBMCs isolated from a cohort of PLWHA. Polychromatic flow cytometry was used to phenotype cells isolated from intestinal biopsies, lymph nodes, and peripheral blood from rhesus macaques and PLWHA. The Alcohol Use Disorders Identification Test (AUDIT) identified hazardous alcohol drinking in PLWHA. Viral load was determined by RT-qPCR and telomere length was measured using qPCR. PBMC CD8+ T-cell activation (CD38+HLA-DR+) and immunosenescence (CD28-) were increased over baseline levels (857% ± 334, p < 0.05; 398% ± 80, p < 0.05, respectively) only in CBA animals not receiving ART. Viral load correlated with CD8+ T-cell immunosenescence in macaque PBMCs (r(s) = 0.49, p = 0.02). Activated immunosenescent T-cell (CD8+CD38+CD28-) frequencies in PBMCs from PLWHA significantly correlated with AUDIT scores (r(s) = 0.75, p = 0.001), while no correlation was observed with CD4+ T-cell and AUDIT scores (r(s) = -0.24, p = 0.38). Activated immunosenescent T-cells had shorter telomeres than CD8+ T-cells (CD8+CD28+) from PLWHA. Our results suggest that CBA and AUD augment immune activation and immunosenescence in SIV-infected macaques and PLWHA.
Asunto(s)
Alcoholismo/inmunología , Consumo Excesivo de Bebidas Alcohólicas/inmunología , Linfocitos T CD8-positivos/efectos de los fármacos , Depresores del Sistema Nervioso Central/farmacología , Etanol/farmacología , Inmunosenescencia/efectos de los fármacos , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Animales , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Infecciones por VIH/inmunología , Inmunosenescencia/inmunología , Intestinos/citología , Intestinos/inmunología , Leucocitos Mononucleares , Ganglios Linfáticos/citología , Ganglios Linfáticos/inmunología , Macaca mulatta , Masculino , ARN Viral/sangre , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Virus de la Inmunodeficiencia de los Simios/genética , Telómero/metabolismo , Carga ViralRESUMEN
Cluster of differentiation molecule (CD)3 and CD28 receptors play crucial roles in T-lymphocyte proliferation. Fe deficiency in man and animals impairs T-lymphocyte proliferation by unknown mechanisms. To test the hypothesis that reduced CD3 and CD28 expression is one of them, thymocytes and splenocytes from control (C; n 24), Fe-deficient (ID; n 24), pair-fed (PF; n 24), and ID mice that were Fe-repleted for 3 (R3; n 24) or 14 d (R14; n 12) were labelled with anti-CD3-fluorescein isothiocyanate and anti-CD28-phycoerythrin antibodies. Positive cells were analysed by flow cytometry. Significant differences were observed among groups in the mean levels of haemoglobin and liver Fe stores (C=PF=R14>R3>ID; P<0.005). While Fe deficiency slightly increased the percentage of CD3+ splenocytes, it reduced that of CD28+ thymocytes in mice with thymus atrophy and splenomegaly (P<0.05). These changes were corrected by Fe repletion. CD28 mean fluorescence intensity (FI) was lower and CD3 FI was higher in lymphocytes from R3 and ID, especially those with splenomegaly, than in those from R14 and PF mice (P<0.05). In vitro Fe chelation by deferoxamine (60 min) significantly decreased CD28 expression (P<0.05), and slightly increased that of CD3 (P>0.05). Spleen cell proliferative responses to concanavalin A and anti-CD3+/-anti-CD28 were reduced by Fe deficiency (ID=R3Asunto(s)
Anemia Ferropénica/inmunología
, Antígenos CD28/inmunología
, Complejo CD3/inmunología
, Linfocitos T/inmunología
, Anemia Ferropénica/metabolismo
, Animales
, Células Cultivadas
, Concanavalina A/farmacología
, Femenino
, Hemoglobinas/metabolismo
, Hierro/metabolismo
, Quelantes del Hierro/farmacología
, Hígado/metabolismo
, Activación de Linfocitos
, Recuento de Linfocitos
, Ratones
, Ratones Endogámicos C57BL
, Mitógenos/farmacología
, Estado Nutricional
, Bazo/citología
, Bazo/inmunología
RESUMEN
The interaction of CD28 and its ligands (CD80, CD86) on antigen presenting cells and that of TCR/CD3-MHC are required for T lymphocyte activation. To determine whether impaired lymphocyte proliferation associated with iron deficiency is due to reduced expression of these ligands, spleen cells obtained from eight to nine C57BL/6 mice/group of iron deficient (ID), iron replete (R), control (C), pair-fed (PF), and high iron (HI) mice were labeled with anti-CD80-fluorescein isothiocyante (FITC) and anti-CD86-FITC. Diets differed only in iron concentration: 5, 50, and 125 mg/kg for the ID, C, and HI, respectively. Mean levels of hemoglobin and liver iron stores of ID and R mice were less than 50% those of C mice (P < 0.005). In non-activated and concanavalin A-treated cultures, significant differences were observed among groups in the percentage of CD80 + cells: ID>R > C = PF = HI (P < 0.05). The same trend was observed for CD86 + cells (P > 0.05). Fluorescence intensity (FI) of either marker did not significantly change by iron status. In vitro iron chelation by deferoxamine (20, 200 microg/ml) for 1, 2, and 24 h increased FI of both markers on unactivated B and T cells (P < 0.05). However, it had no effect on FI of either marker of mitogen-treated cells presumably because the maximum levels are achieved by the mitogen. Lymphocyte proliferative responses to mitogens positively and significantly correlated with CD80 and CD86 FI (r = 0.41-0.59) but negatively correlated with the percentages of CD80 + cells (r = -0.48) (P < 0.05). Data suggest that impaired lymphocyte proliferation associated with iron deficiency is not due to reduced CD80 and CD86 expression.