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1.
Cytokine ; 137: 155299, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33011400

RESUMO

BACKGROUND: Cigarette smoking is a major risk factor for cancer and other diseases. While smoking induces chronic inflammation and aberrant immune responses, the effects of smokeless tobacco products (STPs) on immune responses is less clear. Here we evaluated markers related to immune regulation in smokers (SMK), moist snuff consumers (MSC) and non-tobacco consumers (NTC) to better understand the effects of chronic tobacco use. MATERIALS AND METHODS: Several markers associated with immune regulation were measured in peripheral blood mononuclear cells (PBMCs) from SMK (n = 40), MSC (n = 40), and NTC (n = 40) by flow cytometry. RESULTS: Relative to NTC, seven markers were significantly suppressed in SMK, whereas in MSC, only one marker was significantly suppressed. In a logistic regression model, markers including granzyme B+ lymphocytes, perforin+ lymphocytes, granzyme B+ CD8+T cells, and KLRB1+ CD8+ T cells remained as statistically significant predictors for classifying the three cohorts. Further, cell-surface receptor signaling pathways and cell-cell signaling processes were downregulated in SMK relative to MSC; chemotaxis and LPS-mediated signaling pathways, were upregulated in SMK compared to MSC. A network of the tested markers was constructed to visualize the immunosuppression in SMK relative to MSC. CONCLUSION: Moist snuff consumption is associated with significantly fewer perturbations in inflammation and immune function biomarkers relative to smoking. IMPACT: This work identifies several key immunological biomarkers that differentiate the effects of chronic smoking from the use of moist snuff. Additionally, a molecular basis for aberrant immune responses that could render smokers more susceptible for infections and cancer is provided.


Assuntos
Biomarcadores/sangue , Imunidade , Inflamação/sangue , não Fumantes/estatística & dados numéricos , Fumantes/estatística & dados numéricos , Tabaco sem Fumaça/estatística & dados numéricos , Adulto , Antígenos CD4/sangue , Antígenos CD8/sangue , Quimiocina CCL3/sangue , Estudos de Coortes , Humanos , Inflamação/diagnóstico , Inflamação/imunologia , Leucócitos Mononucleares/metabolismo , Linfócitos/metabolismo , Masculino , Pessoa de Meia-Idade , Subfamília B de Receptores Semelhantes a Lectina de Células NK/sangue , Mapas de Interação de Proteínas , Fatores de Risco , Fator de Necrose Tumoral alfa/sangue
2.
Sci Rep ; 9(1): 13390, 2019 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-31527707

RESUMO

Cigarette smoke-induced chronic inflammation is associated with compromised immune responses. To understand how tobacco products impact immune responses, we assessed transcriptomic profiles in peripheral blood mononuclear cells (PBMCs) pretreated with Whole Smoke-Conditioned Medium (WS-CM) or Smokeless Tobacco Extracts (STE), and stimulated with lipopolysaccharide, phorbol myristate and ionomycin (agonists). Gene expression profiles from PBMCs treated with low equi-nicotine units (0.3 µg/mL) of WS-CM and one high dose of STE (100 µg/mL) were similar to those from untreated controls. Cells treated with medium and high doses of WS-CM (1.0 and 3.0 µg/mL) exhibited significantly different gene expression profiles compared to the low WS-CM dose and STE. Pre-treatment with higher doses of WS-CM inhibited the expression of several pro-inflammatory genes (IFNγ, TNFα, and IL-2), while CSF1-R and IL17RA were upregulated. Pre-treatment with high doses of WS-CM abolished agonist-stimulated secretion of IFNγ, TNF and IL-2 proteins. Pathway analyses revealed that higher doses of WS-CM inhibited NF-ĸB signaling, immune cell differentiation and inflammatory responses, and increased apoptotic pathways. Our results show that pre-treatment of PBMCs with higher doses of WS-CM inhibits immune activation and effector cytokine expression and secretion, resulting in a reduced immune response, whereas STE exerted minimal effects.


Assuntos
Apoptose , Fumar Cigarros/efeitos adversos , Meios de Cultivo Condicionados/farmacologia , Inflamação , Leucócitos Mononucleares/patologia , Nicotina/administração & dosagem , Fumaça/efeitos adversos , Perfilação da Expressão Gênica , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Transdução de Sinais
3.
Data Brief ; 25: 103970, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31297406

RESUMO

This Data in Brief article describes global gene expression profiles from human peripheral blood mononuclear cells (PBMCs) that were treated with preparations from reference combustible and non-combustible tobacco products (TPPs). PBMCs isolated from non-smokers were treated with three non-cytotoxic doses of aqueous preparations from 3R4F cigarettes, termed Whole Smoke-Conditioned Medium (WS-CM) and a single dose of 2S3 moist snuff, termed smokeless tobacco extract (STE). PBMCs were treated with the test articles for 3 hours and the extracted total RNA was reverse transcribed and hybridized to HTA 2.0 Genechip® arrays and scanned using an Affymetrix GeneChip® Scanner 3000. CEL files and CHP files were generated using an Affymetrix Expression console. The CEL files were submitted to the NCBI database with GEO accession number GSE110027. The results of the microarray analyses are found in this Data in Brief article. Ingenuity Pathway Analysis (IPA; Qiagen) was used to conduct core analyses of genes that were differentially expressed by high WS-CM or STE based on the Ingenuity Gene knowledge. Expression of several of the differentially expressed genes was confirmed by RT-PCR. Analyses of these data can be found in the article "Distinct gene expression changes in human peripheral blood mononuclear cells treated with different tobacco product preparations" [1].

4.
Toxicol In Vitro ; 57: 117-125, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30776502

RESUMO

Cigarette smoking exerts diverse physiological effects including immune suppression. To better characterize the biological effects of different categories of tobacco products, a genome-wide gene expression study was performed. Transcriptomic profiling was performed in PBMCs treated with different equi-nicotine units of aqueous extracts of cigarette smoke (termed Whole Smoke-Conditioned Medium, or WS-CM), or a single dose smokeless tobacco extract (STE) prepared from reference tobacco products. WS-CM induced dose-dependent changes in the expression of several genes. No significant expression differences between low WS-CM and media control were detected. However, transcripts were significantly affected by medium WS-CM (479), high WS-CM (2, 703), and STE (2, 156). The overlap between medium WS-CM and STE, and high WS-CM and STE, was minimal (34 and 65 transcripts, respectively). Hierarchical clustering revealed that gene expression profiles for STE and medium WS-CM co-clustered, while those affected by the high dose of WS-CM clustered distinctly. Functional analysis revealed that WS-CM, but not STE, uniquely affected genes involved in immune cell development and inflammatory response. Cascades of upstream regulators (e.g., TNF, IL1ß, NFƙB) were identified for the observed gene expression changes and generally suppressed by WS-CM, but not by STE. Collectively, these findings demonstrate that combustible and non-combustible tobacco products elicit distinct biological effects, which could explain the observed chronic immune suppression in smokers.


Assuntos
Misturas Complexas/toxicidade , Regulação da Expressão Gênica/efeitos dos fármacos , Leucócitos Mononucleares/efeitos dos fármacos , Produtos do Tabaco , Células Cultivadas , Humanos , Leucócitos Mononucleares/metabolismo
5.
Inflammation ; 40(5): 1622-1630, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28577134

RESUMO

Cigarette smoking is a major risk factor for several human diseases. Chronic inflammation, resulting from increased oxidative stress, has been suggested as a mechanism that contributes to the increased susceptibility of smokers to cancer and microbial infections. We have previously shown that whole-smoke conditioned medium (WS-CM) and total particulate matter (TPM) prepared from Kentucky 3R4F reference cigarettes [collectively called as combustible tobacco product preparations (TPPs)] potently suppressed agonist-stimulated cytokine secretion and target cell killing in peripheral blood mononuclear cells (PBMCs). Here we have investigated the role of oxidative stress from TPPs, which alters inflammatory responses in vitro. Particularly, we investigated the mechanisms of WS-CM-induced suppression of select cytokine secretions in Toll-like receptor (TLR) agonist-stimulated cells and target cell killing by effector cells in PBMCs. Pretreatment with N-acetyl cysteine (NAC), a precursor of reduced glutathione and an established anti-oxidant, protected against DNA damage and cytotoxicity caused by exposure to WS-CM. Similarly, secretion of tumor necrosis factor (TNF), interleukin (IL)-6, and IL-8 in response to TLR-4 stimulation was restored by pretreatment with NAC. Target cell killing, a functional measure of cytolytic cells in PBMCs, is suppressed by WS-CM. Pretreatment with NAC restored the target cell killing in WS-CM treated PBMCs. This was accompanied by higher perforin levels in the effector cell populations. Collectively, these data suggest that reducing oxidative stress caused by cigarette smoke components restores select immune responses in this ex vivo model.


Assuntos
Imunidade/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Estresse Oxidativo/imunologia , Produtos do Tabaco/efeitos adversos , Acetilcisteína/farmacologia , Células Cultivadas , Citocinas/metabolismo , Humanos , Leucócitos Mononucleares/citologia , Linfócitos T Citotóxicos/efeitos dos fármacos
6.
BMC Genomics ; 18(1): 156, 2017 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-28193179

RESUMO

BACKGROUND: Among the different tobacco products that are available on the US market, cigarette smoking is shown to be the most harmful and the effects of cigarette smoking have been well studied. US epidemiological studies indicate that non-combustible tobacco products are less harmful than smoking and yet very limited biological and mechanistic information is available on the effects of these alternative tobacco products. For the first time, we characterized gene expression profiling in PBMCs from moist snuff consumers (MSC), compared with that from consumers of cigarettes (SMK) and non-tobacco consumers (NTC). RESULTS: Microarray analysis identified 100 differentially expressed genes (DEGs) between the SMK and NTC groups and 46 DEGs between SMK and MSC groups. However, we found no significant differences in gene expression between MSC and NTC. Both hierarchical clustering and principle component analysis revealed that MSC and NTC expression profiles were more similar than to SMK. Random forest classification identified a subset of DEGs which predicted SMK from either NTC or MSC with high accuracy (AUC 0.98). CONCLUSIONS: PMBC gene expression profiles of NTC and MSC are similar to each other, while SMK exhibit distinct profiles with alterations in immune related pathways. In addition to discovering several biomarkers, these studies support further understanding of the biological effects of different tobacco products. TRIAL REGISTRATION: ClinicalTrials.gov. Identifier: NCT01923402 . Date of Registration: August 14, 2013. Study was retrospectively registered.


Assuntos
Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Fumar , Tabaco sem Fumaça , Transcriptoma , Adulto , Área Sob a Curva , Análise por Conglomerados , Estudos de Coortes , Estudos Transversais , Humanos , Leucócitos Mononucleares/metabolismo , Masculino , Pessoa de Meia-Idade
7.
J Vis Exp ; (95): 52351, 2015 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-25650834

RESUMO

Among other pathophysiological changes, chronic exposure to cigarette smoke causes inflammation and immune suppression, which have been linked to increased susceptibility of smokers to microbial infections and tumor incidence. Ex vivo suppression of receptor-mediated immune responses in human peripheral blood mononuclear cells (PBMCs) treated with smoke constituents is an attractive approach to study mechanisms and evaluate the likely long-term effects of exposure to tobacco products. Here, we optimized methods to perform ex vivo assays using PBMCs stimulated by bacterial lipopolysaccharide, a Toll-like receptor-4 ligand. The effects of whole smoke-conditioned medium (WS-CM), a combustible tobacco product preparation (TPP), and nicotine were investigated on cytokine secretion and target cell killing by PBMCs in the ex vivo assays. We show that secreted cytokines IFN-γ, TNF, IL-10, IL-6, and IL-8 and intracellular cytokines IFN-γ, TNF-α, and MIP-1α were suppressed in WS-CM-exposed PBMCs. The cytolytic function of effector PBMCs, as determined by a K562 target cell killing assay was also reduced by exposure to WS-CM; nicotine was minimally effective in these assays. In summary, we present a set of improved assays to evaluate the effects of TPPs in ex vivo assays, and these methods could be readily adapted for testing other products of interest.


Assuntos
Leucócitos Mononucleares/efeitos dos fármacos , Nicotiana/química , Nicotiana/toxicidade , Fumaça/efeitos adversos , Adulto , Linhagem Celular , Citocinas/imunologia , Humanos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/imunologia , Nicotina/farmacologia , Material Particulado/química , Material Particulado/toxicidade , Alvéolos Pulmonares/citologia , Alvéolos Pulmonares/efeitos dos fármacos , Alvéolos Pulmonares/imunologia , Fumaça/análise
8.
Toxicol Appl Pharmacol ; 279(2): 211-9, 2014 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-24927667

RESUMO

Skin fibroblasts comprise the first barrier of defense against wounds, and tobacco products directly contact the oral cavity. Cultured human dermal fibroblasts were exposed to smokeless tobacco extract (STE), total particulate matter (TPM) from tobacco smoke, or nicotine at concentrations comparable to those found in these extracts for 1h or 5h. Differences were identified in pathway-specific genes between treatments and vehicle using qRT-PCR. At 1h, IL1α was suppressed significantly by TPM and less significantly by STE. Neither FOS nor JUN was suppressed at 1h by tobacco products. IL8, TNFα, VCAM1, and NFκB1 were suppressed after 5h with STE, whereas only TNFα and NFκB1 were suppressed by TPM. At 1h with TPM, secreted levels of IL10 and TNFα were increased. Potentially confounding effects of nicotine were exemplified by genes such as ATF3 (5h), which was increased by nicotine but suppressed by other components of STE. Within 2h, TPM stimulated nitric oxide production, and both STE and TPM increased reactive oxygen species. The biological significance of these findings and utilization of the gene expression changes reported herein regarding effects of the tobacco product preparations on dermal fibroblasts will require additional research.


Assuntos
Dermatite/etiologia , Fibroblastos/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Nicotina/toxicidade , Agonistas Nicotínicos/toxicidade , Pele/efeitos dos fármacos , Produtos do Tabaco/toxicidade , Poluição por Fumaça de Tabaco/efeitos adversos , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Dermatite/genética , Dermatite/imunologia , Dermatite/metabolismo , Fibroblastos/imunologia , Fibroblastos/metabolismo , Genes Precoces , Humanos , Mediadores da Inflamação/metabolismo , Óxido Nítrico/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , RNA Mensageiro/metabolismo , Pele/imunologia , Pele/metabolismo , Superóxidos/metabolismo , Fatores de Tempo
9.
Toxicol In Vitro ; 27(6): 1992-2004, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23851003

RESUMO

Natural killer (NK) cells and T cells play essential roles in innate and adaptive immune responses in protecting against microbial infections and in tumor surveillance. Although evidence suggests that smoking causes immunosuppression, there is limited information whether the use of smokeless tobacco (ST) products affects immune responses. In this study, we assessed the effects of two preparations of cigarette smoke, ST extract and nicotine on T cell and NK cell responses using Toll-like receptor-ligand stimulated human peripheral blood mononuclear cells (PBMCs). The tobacco product preparations (TPPs) tested included whole smoke conditioned media (WS-CM), total particulate matter (TPM) and a ST product preparation in complete artificial saliva (ST/CAS). The PBMCs were stimulated with polyinosinic:polycytidylic acid (poly I:C) and lipopolysaccharide (LPS). A marked reduction of the expression of intracellular IFN-γ and TNF-α was evident in NK cells and T cells treated with WS-CM and TPM. Consistently, attenuation of ligand-induced secretion of cytokines (IL-1ß, IL-10, IL-12 and TNF-α) from PBMCs treated with WS-CM and TPM were observed. While the treatment with TPPs did not alter the expression of the maturation marker CD69, WS-CM and TPM inhibited the cytolytic activity of human PBMCs. Suppression of perforin by WS-CM was also detected. Although interference from the vehicle confounded the interpretation of effects of ST/CAS, some effects were evident only at high concentrations. Nicotine treatment minimally impacted expression of cytokines and cytolytic activity. Data presented herein suggests that the function of NK cells and T cells is influenced by exposure to TPPs (based on equi-nicotine units) in the following order: WS-CM>TPM>ST/CAS. These findings are consistent with the hypothesis put forward by others that chronic smoking leads to immunosuppression, an effect that may contribute to increased microbial infections and cancer incidence among smokers.


Assuntos
Células Matadoras Naturais/efeitos dos fármacos , Leucócitos Mononucleares/efeitos dos fármacos , Nicotina/toxicidade , Fumaça/efeitos adversos , Linfócitos T/efeitos dos fármacos , Produtos do Tabaco/toxicidade , Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos T/imunologia , Células Cultivadas , Citocinas/imunologia , Humanos , Células K562 , Células Matadoras Naturais/imunologia , Lectinas Tipo C/imunologia , Leucócitos Mononucleares/imunologia , Lipopolissacarídeos , Perforina/imunologia , Poli I-C , Saliva , Linfócitos T/imunologia
10.
Toxicol Sci ; 134(1): 18-25, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23629517

RESUMO

Complete artificial saliva (CAS) is a saliva substitute often used as a vehicle for test articles, including smokeless tobacco products. In the course of a study employing normal adult human dermal fibroblasts (HDFa) as a model in vitro, we discovered that CAS as a vehicle introduced a significant change in the expression of proinflammatory cytokines. To determine the effects of CAS on gene expression, real-time quantitative reverse-transcriptase PCR gene array analysis was used. Results indicate that robust changes in the expression of the proinflammatory cytokine interleukin 8 (IL8) and the vascular cell adhesion molecule 1 (VCAM1) occur within 5h of exposure to CAS. To determine whether CAS also alters cytokine release into the culture media, cytometric bead array assays for human inflammatory cytokines were performed. Analysis shows that CAS induced the release of IL8 and IL6. This study focused on determining which components in CAS were responsible for the proinflammatory response in HDFa. The following components were investigated: α-amylase, lysozyme, acid phosphatase, and urea. Results demonstrated that enzymatically active α-amylase induced gene expression for proinflammatory cytokines IL8, IL6, tumor necrosis factor-α, and IL1α and for VCAM1. Therefore, it is important to carefully evaluate the "vehicle effects" of CAS and its components in in vitro toxicology research.


Assuntos
Citocinas/genética , Fibroblastos/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Saliva Artificial/toxicidade , alfa-Amilases/toxicidade , Células Cultivadas , Meios de Cultura/química , Citocinas/imunologia , Citocinas/metabolismo , Fibroblastos/imunologia , Humanos , Interleucina-1alfa/genética , Interleucina-1alfa/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Saliva Artificial/química , Pele/citologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Molécula 1 de Adesão de Célula Vascular/genética , alfa-Amilases/análise , alfa-Amilases/metabolismo
11.
Regul Toxicol Pharmacol ; 64(3): 350-60, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22996032

RESUMO

Acute exposure to cigarette smoke or its components triggers diverse cellular effects, including cytotoxicity. However, available data regarding the potential cytotoxic effects of smokeless tobacco (ST) extracts lack consensus. Here, we investigated the relative biological effects of 2S3 reference ST, and whether ST elicits differential cellular/molecular responses compared to combustible tobacco product preparations (TPPs) prepared from 3R4F cigarettes. Total particulate matter (TPM) and whole smoke conditioned medium (WS-CM) were employed as combustible TPPs, while the ST extract was used as non-combustible TPP. HL60, THP1 cells and human PBMCs were used to examine the effects of TPPs in short-term cell culture. Corresponding EC(50) values, normalized for nicotine content of the TPPs, suggest that combustible TPPs induced higher cytotoxicity as follows: WS-CM TPM ≥ ≫ST extract>nicotine. While all three TPPs induced detectable levels of DNA damage and IL8 secretion, the combustible TPPs were significantly more potent than the ST preparation. The major PBMC subsets showed differential cytotoxicity to combustible TPPs as follows: CD4>CD8>monocytes>NK cells. These findings suggest that, relative cytotoxic and other cell biological effects of TPPs are dose-dependent, and that ST extract is the least cytotoxic TPP tested in this study.


Assuntos
Nicotina/toxicidade , Material Particulado/toxicidade , Produtos do Tabaco/toxicidade , Tabaco sem Fumaça/toxicidade , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/patologia , Dano ao DNA/efeitos dos fármacos , Células HL-60 , Humanos , Interleucina-8/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/patologia , Macrófagos/efeitos dos fármacos , Macrófagos/patologia , Monócitos/efeitos dos fármacos , Monócitos/patologia , Fumaça/efeitos adversos
12.
J Virol ; 84(22): 12093-8, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20844045

RESUMO

Vesicular stomatitis viruses (VSVs) containing wild-type (wt) or mutant matrix (M) proteins are being developed as candidate vaccine vectors due to their ability to induce innate and adaptive immunity. Viruses with wt M protein, such as recombinant wild-type (rwt) virus, stimulate maturation of dendritic cells (DC) through Toll-like receptor 7 (TLR7) and its adaptor molecule MyD88. However, M protein mutant viruses, such as rM51R-M virus, stimulate both TLR7-positive and TLR7-negative DC subsets. The goal of this study was to determine whether the ability of rwt and rM51R-M viruses to induce maturation of human DC can be enhanced by engineering these vectors to express bacterial flagellin. Flagellin expressed from the rwt virus genome partially protected human DC from VSV-induced shutoff of host protein synthesis and promoted the production of interleukin 6 (IL-6) and IL-1ß. In addition, DC infected with rwt virus expressing flagellin were more effective at stimulating gamma interferon (IFN-γ) production from CD8(+) allogeneic T cells than DC infected with rwt virus. Although rM51R-M virus effectively stimulated human DC, flagellin expressed from the rM51R-M virus genome enhanced the production of cytokines. Furthermore, mice immunized with both rwt and rM51R-M viruses expressing flagellin had enhanced anti-VSV antibody responses in vivo. Therefore, rwt and rM51R-M viruses expressing flagellin may be promising vectors for the delivery of foreign antigen due to their potential to stimulate DC function.


Assuntos
Células Dendríticas/imunologia , Flagelina/genética , Engenharia Genética , Estomatite Vesicular/imunologia , Vírus da Estomatite Vesicular Indiana/imunologia , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/imunologia , Animais , Linhagem Celular , Células Cultivadas , Células Dendríticas/virologia , Feminino , Flagelina/imunologia , Humanos , Masculino , Camundongos , Mutação , Salmonella enterica/genética , Salmonella enterica/imunologia , Estomatite Vesicular/virologia , Vírus da Estomatite Vesicular Indiana/genética
13.
Virology ; 404(2): 225-30, 2010 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-20627346

RESUMO

It has become clear that T cells with the potential to negatively regulate the immune response are normal constituents of the immune system. These cells often mediate their effects through the production of immunosuppressive factors. At present our understanding of how these cells are generated is limited. Here we report the presence of a population of IL-10-producing, virus-specific CD8+ T cells in the lungs of mice following acute respiratory infection. These cells were only found at minimal levels in the spleen and draining lymph node; instead they were restricted primarily to the infected lung tissue. A major finding from this study is demonstration that the ability to produce IL-10 can be acquired by IFNgamma-producing effector cells following entry into the infected lung. These studies suggest IL-10 production is the result of further differentiation of an antigen-specific CD8+ T cell that is governed by signals present in infected lung tissue.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Interferon gama/metabolismo , Interleucina-10/metabolismo , Pulmão/citologia , Vírus da Parainfluenza 5 , Infecções por Rubulavirus/imunologia , Transferência Adotiva , Animais , Antígenos Virais , Regulação da Expressão Gênica , Pulmão/imunologia , Linfonodos/citologia , Camundongos , Camundongos Endogâmicos BALB C , Baço/citologia
14.
J Leukoc Biol ; 87(6): 977-88, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20103768

RESUMO

CD8(+) T cells play a critical role in the clearance of respiratory pathogens. Thus, it is surprising that functional inactivation of lung effectors has been observed in many models of viral infection. Currently, the molecular defect responsible for the shut-off of function in these cells is unknown. In the present study, we addressed this question using a model of respiratory infection with the paramyxovirus SV5. Nonfunctional cells were found to exhibit decreases in SOCE, resulting in reduced NFAT1 activation. Notably, function could be restored by the provision of increased levels of extracellular calcium. The reduced ability to mobilize calcium was associated with reduced expression of ORAI1, the CRAC channel subunit. These findings reveal a previously unknown mechanism for the negative regulation of function in effector T cells.


Assuntos
Linfócitos T CD8-Positivos/metabolismo , Canais de Cálcio/metabolismo , Sinalização do Cálcio , Cálcio/metabolismo , Pulmão/metabolismo , Animais , Western Blotting , Canais de Cálcio/genética , Células Cultivadas , Citocinas/metabolismo , Eletrofisiologia , Feminino , Imunização , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Fatores de Transcrição NFATC/genética , Fatores de Transcrição NFATC/metabolismo , Proteína ORAI1 , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Molécula 1 de Interação Estromal
15.
J Virol ; 82(22): 10975-85, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18787007

RESUMO

The paramyxovirus simian virus 5 (SV5) is a poor activator of human dendritic cell (DC) maturation pathways in vitro, and infected DC do not upregulate cell surface costimulatory proteins or secretion of immunomodulatory cytokines. We evaluated the hypothesis that activation of SV5-infected DC would be enhanced by engineering SV5 to express a Toll-like-receptor (TLR) ligand. To test this hypothesis, a novel virus was engineered such that the gene encoding an intracellular form of the TLR5 ligand flagellin was expressed from the genome of wild-type (WT) SV5 (SV5-flagellin). Cells infected in vitro with the flagellin-expressing virus released low levels of biologically active flagellin, which was capable of stimulating TLR5 signaling. Infection of human peripheral blood mononuclear cell-derived immature DC with SV5-flagellin resulted in enhanced levels of interleukin-6 (IL-6) and IL-12 compared to infection with DC with the parental virus, WT SV5. In contrast to cytokine induction, the flagellin-expressing virus did not appreciably increase DC surface expression of the costimulatory molecule CD80 or CD86 above the level seen with WT SV5 alone. In mixed-culture assays, DC infected with the flagellin-expressing virus were more effective at activating gamma interferon secretion from both CD8(+) and CD4(+) allogeneic T cells than DC infected with WT SV5. Our results with SV5-directed intracellular expression of flagellin may be applicable to other vectors or pathogenic viruses where overcoming impairment of DC activation could contribute to the development of safer and more effective vaccines.


Assuntos
Adjuvantes Imunológicos/farmacologia , Células Dendríticas/imunologia , Flagelina/imunologia , Flagelina/farmacologia , Vírus da Parainfluenza 5/imunologia , Receptor 5 Toll-Like/imunologia , Adjuvantes Imunológicos/genética , Antígeno B7-1/biossíntese , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Células Cultivadas , Flagelina/genética , Flagelina/metabolismo , Humanos , Interleucina-12/metabolismo , Interleucina-6/metabolismo , Vírus da Parainfluenza 5/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Recombinação Genética , Receptor 5 Toll-Like/metabolismo
16.
J Leukoc Biol ; 83(3): 564-74, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18079210

RESUMO

Recently, several studies, including those with respiratory syncytial virus, mouse pneumovirus, and simian virus 5, have reported that virus-specific CD8+ effector cells entering the lung as a result of respiratory infection undergo significant loss of function. The impaired function in these cells has been proposed to be the result of infection-induced changes in the lung. Although virus-specific effects may contribute to regulation of T cells in the lung, the findings from this study provide evidence that the basal lung environment is sufficient to promote loss of function in effector cells. Loss of function occurs within 48 h of entry into the lung and is most evident in cells residing in the lung parenchyma. These findings suggest an additional paradigm for the immunoregulation of effector cells that enter the lung as a result of virus infection.


Assuntos
Pneumopatias/virologia , Pulmão/imunologia , Linfócitos T/imunologia , Vacínia/imunologia , Viroses/imunologia , Transferência Adotiva , Animais , Apoptose , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Feminino , Pulmão/virologia , Pneumopatias/imunologia , Pneumopatias/patologia , Camundongos , Camundongos Endogâmicos BALB C , Baço/imunologia , Baço/virologia , Viroses/patologia
17.
Virology ; 365(1): 144-56, 2007 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-17459446

RESUMO

Infection of primary cultures of human immature monocyte-derived dendritic cells (moDC) with the paramyxovirus Simian Virus 5 (SV5) results in extensive cytopathic effect (CPE) and induction of apoptosis, but DC maturation pathways are not activated. In this study, we investigated the relationship between SV5-induced apoptosis and the lack of DC maturation. Reducing CPE and apoptosis in SV5-infected immature DC by the addition of a pancaspase inhibitor resulted in only low level expression of maturation markers CD40, CD80 and CD86, suggesting that SV5 infection either actively blocked maturation pathways or failed to provide sufficient signals to activate maturation. To distinguish between these hypotheses, SV5-infected immature DC were challenged with agonists that stimulate toll-like receptors (TLRs). Treatment with the TLR-4 agonist LPS or TLR-6 agonist FSL1 enhanced cell surface expression of CD40, CD80 and CD86 on SV5-infected cells to levels approaching that of mock-infected TLR-treated moDC, but treatment with agonists for TLR-2, -3, -5 or -8 had little effect. Addition of TLR-4 or -6 agonists to SV5-infected DC also dramatically reduced CPE and apoptosis, but the levels of viral protein and virus yield were not affected. Similarly, SV5-infected immature moDC were matured by treatment with IL-1beta, and these mature infected cells also showed reduced CPE and apoptosis. In the presence of NFkB inhibitors, TLR-4 and -6 agonists did not promote maturation or reduce apoptosis of SV5-infected DC, indicating that maturation and cell survival were both dependent on signaling through NFkB-dependent pathways. Our results suggest a model whereby SV5 replication induces apoptosis in immature DC but fails to provide strong maturation signals, while activation of NFkB-dependent pathways by exogenous ligands can lead to moDC maturation and override SV5-induced cell death.


Assuntos
Apoptose , Células Dendríticas/virologia , NF-kappa B/metabolismo , Vírus da Parainfluenza 5/fisiologia , Receptor 4 Toll-Like/agonistas , Receptor 6 Toll-Like/agonistas , Células Dendríticas/fisiologia , Humanos , Sistema de Sinalização das MAP Quinases/fisiologia , Vírus da Parainfluenza 5/genética , Receptores Toll-Like/metabolismo
18.
J Virol ; 80(7): 3416-27, 2006 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16537609

RESUMO

Human epithelial cells infected with the parainfluenza virus simian virus 5 (SV5) show minimal activation of host cell interferon (IFN), cytokine, and cell death pathways. In contrast, a recombinant SV5 P/V gene mutant (rSV5-P/V-CPI-) overexpresses viral gene products and is a potent inducer of IFN, proinflammatory cytokines, and apoptosis in these cells. In this study, we have compared the outcomes of wild-type (WT) SV5 and rSV5-P/V-CPI- infections of primary human dendritic cells (DC), important antigen-presenting cells for initiating adaptive immune responses. We have tested the hypothesis that a P/V mutant which activates host antiviral responses will be a more potent inducer of DC maturation and function than WT rSV5, which suppresses host cell responses. Infection of peripheral blood mononuclear cell-derived immature DC with WT rSV5 resulted in high levels of viral protein and progeny virus but very little increase in cell surface costimulatory molecules or secretion of IFN and proinflammatory cytokines. In contrast, immature DC infected with the rSV5-P/V-CPI- mutant produced only low levels of viral protein and progeny virus, but these infected cells were induced to secrete IFN-alpha and other cytokines and showed elevated levels of maturation markers. Unexpectedly, DC infected with WT rSV5 showed extensive cytopathic effects and increased levels of active caspase-3, while infection of DC with the P/V mutant was largely noncytopathic. In mixed-culture assays, WT rSV5-infected DC were impaired in the ability to stimulate proliferation of autologous CD4+ T cells, whereas DC infected with the P/V mutant were very effective at activating T-cell proliferation. The addition of a pancaspase inhibitor to DC infected with WT rSV5 reduced cytopathic effects and resulted in higher surface expression levels of maturation markers. Our finding that the SV5 P/V mutant has both a reduced cytopathic effect in human DC compared to WT SV5 and an enhanced ability to induce DC function has implications for the rational design of novel recombinant paramyxovirus vectors based on engineered mutations in the viral P/V gene.


Assuntos
Células Dendríticas/virologia , Mutação , Vírus da Parainfluenza 5/genética , Vírus da Parainfluenza 5/patogenicidade , Fosfoproteínas/fisiologia , Proteínas Virais/fisiologia , Proteínas Estruturais Virais/fisiologia , Apoptose , Western Blotting , Caspase 3 , Caspases/análise , Proliferação de Células , Células Cultivadas , Efeito Citopatogênico Viral , Células Dendríticas/citologia , Células Dendríticas/enzimologia , Ativação Enzimática , Corantes Fluorescentes , Proteínas de Fluorescência Verde/metabolismo , Humanos , Imuno-Histoquímica , Indóis , Interferon Tipo I/metabolismo , Microscopia de Fluorescência , Vírus da Parainfluenza 5/crescimento & desenvolvimento , Fosfoproteínas/biossíntese , Fosfoproteínas/genética , Proteínas de Ligação a RNA , Recombinação Genética , Fator de Transcrição STAT1/metabolismo , Linfócitos T/fisiologia , Proteínas Virais/biossíntese , Proteínas Virais/genética , Proteínas Estruturais Virais/biossíntese , Proteínas Estruturais Virais/genética
19.
J Virol ; 79(6): 3339-49, 2005 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15731228

RESUMO

For many respiratory pathogens, CD8+ T cells have been shown to play a critical role in clearance. However, there are still many unanswered questions with regard to the factors that promote the most efficacious immune response and the potential for immunoregulation of effector cells at the local site of infection. We have used infection of the respiratory tract with the model paramyxovirus simian virus 5 (SV5) to study CD8+ T-cell responses in the lung. For the present study, we report that over time a population of nonresponsive, virus-specific CD8+ T cells emerged in the lung, culminating in a lack of function in approximately 85% of cells specific for the immunodominant epitope from the viral matrix (M) protein by day 40 postinfection. Concurrent with the induction of nonresponsiveness, virus-specific cells that retained function at later times postinfection exhibited an increased requirement for CD8 engagement. This change was coupled with a nearly complete loss of functional phosphoprotein-specific cells, a response previously shown to be almost exclusively CD8 independent. These studies add to the growing evidence for immune dysregulation following viral infection of the respiratory tract.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Pulmão/imunologia , Vírus da Parainfluenza 5/imunologia , Infecções Respiratórias/imunologia , Infecções Respiratórias/virologia , Infecções por Rubulavirus/imunologia , Animais , Antígenos Virais/imunologia , Modelos Animais de Doenças , Epitopos/imunologia , Feminino , Interferon gama/biossíntese , Pulmão/patologia , Linfonodos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Fosfoproteínas/imunologia , Infecções Respiratórias/patologia , Infecções por Rubulavirus/patologia , Infecções por Rubulavirus/virologia , Proteínas da Matriz Viral/imunologia , Proteínas Virais/imunologia
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