Differences in biomarkers of inflammation and immune responses in chronic smokers and moist snuff users.

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.

Gene expression profiles associated with cigarette smoking and moist snuff consumption.

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.

Regulation of gene expression by tobacco product preparations in cultured human dermal fibroblasts.

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.

Evaluation of cytotoxicity of different tobacco product preparations.

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.

Stimulation of human dendritic cells by wild-type and M protein mutant vesicular stomatitis viruses engineered to express bacterial flagellin.

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.

Engineered expression of the TLR5 ligand flagellin enhances paramyxovirus activation of human dendritic cell function.

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.

Loss of function in virus-specific lung effector T cells is independent of infection.

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.

Global gene expression profiles from PBMCs treated with reference tobacco product preparations.

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].

Cigarette smoke preparations, not moist snuff, impair expression of genes involved in immune signaling and cytolytic functions.

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.

Distinct gene expression changes in human peripheral blood mononuclear cells treated with different tobacco product preparations.

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.