Elsevier

Immunobiology

Volume 222, Issue 2, February 2017, Pages 169-175
Immunobiology

Cigarette smoking represses expression of cytokine IL-12 and its regulator miR-21—An observational study in patients with coronary artery disease

https://doi.org/10.1016/j.imbio.2016.10.007Get rights and content

Abstract

Rationale

The heterodimer IL-12 is an inducer of Th1 responses and stimulates INFƴ production. Micro-RNA-21 (miR-21) is described as a key regulator of the pro-inflammatory response and has IL-12p35 mRNA as one of its main targets. The IL-12p40 1188A/C genetic variant located in 3’untranslated region (UTR), thus environmentally exposed, has further been reported to modify IL-12 levels. We have previously reported on the lowering effect of cigarette smoke on circulating IL-12 in patients with coronary artery disease (CAD).

Objectives

To explore if cigarette smoking affects IL-12p35, IL-12p40, INFƴ and miR-21 gene-expression and further modulates any effect of the IL-12p40 polymorphism on circulating IL-12 levels.

Methods and Results

The IL-12p40 1188A/C polymorphism was analyzed in 1001 stable CAD patients, of which 330 subjects were included for IL-12p35, IL-12p40 and INFƴ gene-expression analyses in circulating leukocytes and 200 were further selected for plasma miR-21 analysis.

Smoking associated with lower expression of miR-21 and its target IL-12p35 mRNA (adjusted p < 0.05, both) whereas the influence on INFƴ expression tended to be high-dose reliant (p = 0.057). The IL-12p40 CC genotype associated with elevated circulating IL-12 levels, however, when stratified according to smoking, only in the non-smoking group (adjusted p < 0.05).

Although the markers were mainly downregulated in current smokers, their inter-correlations were potentiated.

Conclusion

Smoking associated with reduced miR-21 gene-repression and the results can therefore not explain the previously observed reduction in circulating IL-12. Smoking attenuated the IL-12 pro-inflammatory axis in which the investigated IL-12p40 genetic variant may have different clinical impact in smokers vs non-smokers.

Introduction

The pro-inflammatory cytokine interleukin (IL)-12 is critical in the initiation and progression of the Th-1 response, characterized by production of interferon gamma (INFƴ) by different T-cells and natural killer cells (Trinchieri and Scott, 1995). IL-12 secretion occurs early in the immune response and seems to play a key role in the regulation of innate and adaptive immunity and to mediate anti-tumor activity (Trinchieri and Scott, 1995, Tahara and Lotze, 1995). The heterodimer IL-12p70 (70 kDa (kilo Daltons)), henceforth referred to as IL-12, is composed of two disulfide-linked polypeptide chains, IL-12p35 (35 kDa) and IL-12p40 (40 kDa), synthesized from two differently located genes, which are transcriptionally and independently regulated (Sieburth et al., 1992). IL-12 is mainly produced by B-cells, monocytes, macrophages and dendritic cells (DCs) and the release of bioactive IL-12 is thought to require that both subunits are expressed and further dimerized within the same cell (Trinchieri, 2003).

We have previously reported on the substantial lowering effect of cigarette smoking on circulating IL-12 levels in patients with coronary artery disease (CAD) (Opstad et al., 2016). Similar effects have been observed in bronchoalveolar lavage and gingival crevicular fluid of current smokers (Burnham et al., 2013, Tymkiw et al., 2011). In vitro studies have reported decreased IL-12p40 mRNA levels in macrophages exposed to cigarette smoke extract (CSE), probably mediated by enhanced levels of the p40 transcriptional repressor c-fos (Kroening et al., 2008). Independent of cell-type or species origin, exposure of CSE has further been described in in vitro studies to modify INFƴ expression and to impair INFƴ signaling, although contradictory reported (Modestou et al., 2010, Lee et al., 2014, Lugade et al., 2014, Dhillon et al., 2009).

MicroRNAs (miRNAs) are naturally occurring short non-coding RNA molecules that regulate the expression of target genes post-transcriptionally, by messenger RNA (mRNA) degradation and/or translational inhibition. MiRNAs can be released by cells into the circulation in association with micro-particles, exosomes, or specific proteins, thus serving as potential biomarkers. MiRNA-21 (MiR-21) is the most abundantly expressed small non-coding RNA in multiple mammalian cells, including hematopoietic cells of the immune system, like B- and T-cells, monocytes, macrophages and DCs. It is upregulated in many disease states as cardiac injury, multiple neoplasms and inflamed tissue (Sheedy, 2015). Targets of miR-21 are mainly tumor suppressor genes, including the IL-12p35 gene, in which a functional, conserved miR-21 binding site in the 3’untranslated region (UTR) has been described (Lu et al., 2009). Repression of IL-12p35 expression by miR-21 could therefore lead to less production of IL-12. MiR-21 has further been defined as the major regulator of Th1 versus Th2 responses, thus influencing the IL-12/INFƴ pro-inflammatory pathway (Lu et al., 2011).

Genetic variability of the IL-12 genes may also influence the extent of IL-12 production. Many studies have focused on the polymorphic site at position 1188 in the 3′ UTR of the IL-12p40 gene, which was reported to increase in vitro IL-12 secretion rather than to increase levels of the single IL-12p40 subunit (Seegers et al., 2002). The 1188 C-allele, also associated with decreased IL-12p40 serum levels (Wang et al., 2013), has been reported to increase the overall risk of cancers (Zhou et al., 2012) and was further associated with increased risk of bladder cancer in cigarette smokers (Ebadi et al., 2014) indicating a IL-12p40 gene-environment interaction.

According to our previous findings of low IL-12 levels in smokers, we aimed in the present study to investigate the influence of cigarette smoke on markers involved in the IL-12/INFƴ pro-inflammatory axis, including circulating miR-21 and IL-12p35, IL-12p40 and INF-γ gene-expression in circulating leukocytes, in patients with stable CAD. The IL-12p40 1188 A/C polymorphism was further analyzed according to IL-12 levels and smoking habits. We hypothesized an up-regulation of miR-21 and repression of the IL-12 and INFƴ genes in current smokers, with potential influence of the IL-12 p40 genetic variant.

Section snippets

Subjects

The present investigation is a sub-study of the ASCET trial in Norway (Pettersen et al., 2004) (Pettersen et al., 2012), which included 1001 patients with stable CAD (97% Caucasians, 78% men, 20% current smokers, mean age 62 year) in the time period from 2003 to 2008. Smoking habits were recorded in 4 categories: 0; non-smokers, 1; quitted smoking > 3 months ago, 2; current smokers  10 cigarettes per day, 3; current smokers > 10 cigarettes per day. The data analyses are mainly based on differences

Results

Demographic data in the miR-21 cohort (n = 200) according to cigarette smoking are presented in Table 1, showing similar distribution of the variables between current smokers and non-smokers, except of higher CRP values in the smoking group. We have previously reported on the lowering effect of smoking on circulating IL-12 in the total ASCET population (n = 1001) (Opstad et al., 2016). In the present miR-21 cohort, circulating IL-12 was similarly and significantly lower (43%) in current smokers vs.

Discussion

Our hypothesis of increased miR-21 expression in current smokers could have explained the previously reported lower circulating IL-12 levels in these subjects. However, cigarette smoking was significantly associated with lower expression of miR-21 (Fig. 1A) and its target IL-12p35 mRNA (Fig. 2A) in the present study. Hence, our miR-21 results cannot clarify the pronounced reduction in circulating IL-12 in current smokers, although in a small group of patients with particular low IL-12 levels,

Conclusion

In conclusion, circulating mir-21 and expression of the IL-12p35 gene were observed significantly downregulated in current smokers with CAD. The results may indicate miR-21 as a modest regulator of IL-12p35mRNA, as the variables seem to mirror each other rather than being inversely correlated, hence the nexus of cause and consequence needs to be explored. The IL-12/INFƴ pro-inflammatory axis seemed further to be potentiated, although dampened, by cigarette smoking, suggesting an altered

Conflict of interest

None.

Source of funding

The work was funded by the Norwegian Council for Cardiovascular Diseases and Stein Erik Hagen Foundation for Clinical Heart Research, Oslo Norway. Theses funding sources were not involved in the research and preparation of the article.

Acknowledgments

Vibeke Bratseth and Sissel Åkra are acknowledged for blood sampling handling in the ASCET trial.

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