Vaccination, a human model of inflammation, activates systemic inflammation but does not trigger proinflammatory gene expression in adipose tissue

Dear Sir, Inflammation plays a key role in the development of atherosclerosis [1]. Many studies have emphasized inflammatory markers as prognostic for atherosclerosis and myocardial infarction [2, 3]. One of these, interleukin-6 (IL-6), is a multifunctional cytokine with an important role in host defence. It is involved in the development of cells and tissues as well as in different pathological conditions. IL-6 is not constitutively produced but can be synthesized in response to inflammatory stimuli such as interleukin-1β (IL-1β), lipopolysaccharide and tumour necrosis factor-alpha (TNF-α) [4, 5]. IL-6 is produced by many different cell types including monocytes/macrophages, fibroblasts, endothelial cells and adipocytes. It regulates production of adhesion molecules involved in the release of other cytokines and induces the hepatic synthesis of C-reactive protein (CRP). In vivo release of IL-6 from human adipose tissue (AT) has been demonstrated [6]. Furthermore, positive associations between IL-6 and TNF-α in AT and circulating CRP have been shown [7]. TNF-α is synthesized by cells of the immune system and it is a strong mediator of inflammatory and immune functions [5]. Furthermore, it is known to regulate growth and differentiation of many different cell types. Macrophages in AT can produce TNF-α shown to be associated with proinflammatory activity that may contribute to atherosclerosis [8]. IL-1β is produced by several cell types including macrophages and it is a potent inducer of fever and the acute phase response [5]. The positive correlation seen between proinflammatory cytokines in AT and circulating CRP is of particular interest as it has been shown that decreased AT macrophage infiltration was associated with an improved inflammatory profile following weight loss in obese subjects [9]. Moreover, a positive association between body mass index and death from cardiovascular disease has been demonstrated [10], indicating that AT proinflammatory cytokines are pathogenic in atherosclerosis. To elucidate a possible connection between systemic inflammation and inflammatory activity in AT in humans, we investigated whether a standardized inflammatory stimulus activates AT and circulating peripheral blood mononuclear cells (PBMC). To standardize inflammation, we used a model of vaccination against Salmonella typhi. Current knowledge about stimulated inflammatory activity in different cell types and tissues is mostly based on animal and in vitro studies, therefore it is of particular interest to investigate this in humans. Our hypothesis was that an acute systemic inflammation would stimulate inflammatory activity in AT that could sustain the systemic inflammatory response. Eighteen healthy volunteers (16 men and two postmenopausal women) who had participated in two previous studies were invited (Table 1). Every second individual was allocated to the vaccine or control group. There have been conflicting data as to whether genetic variation in the IL-6 gene could explain variability in stimulated plasma levels of IL-6 [11–13]. Results from our group have clearly demonstrated that the G-allele of the 174 G>C polymorphism in the promoter region of the IL-6 gene is associated with increased IL-6 levels in plasma after vaccination [14]. To avoid differences in inflammatory response due to this polymorphism all subjects in this study were homozygous for the common −174 G allele. All subjects gave informed written consent for participation in the study which was approved by the Ethics Committee of the Karolinska Institutet. Venous blood samples were obtained after 0, 4, 8, 12 and 24 h. After the initial blood sample, subjects in the vaccine group received an injection with vaccine against S. typhi (Typhim Vi; Sanofi Pasteur MSD, Solna, Sweden), 0.5 mL (25 μg), intramuscularly in the left shoulder. Four hours after the first blood sampling all subjects underwent a subcutaneous fat biopsy from the periumbilical area of the abdomen, as described previously[15]. The biopsy of 300–500 mg was washed in saline and immediately frozen in RNAlater (Ambion, Inc., Austin, TX, USA) at −70°C for gene expression studies. At 0 and 4 h venous blood samples were obtained for analysis of gene expression. These blood samples were taken in cell preparation tubes (BD, Franklin Lakes, NJ, USA) for separation of PBMC from whole blood. IL-6 in EDTA plasma was analysed by a high sensitivity enzyme immunoassay (R&D Systems Inc., Minneapolis, MN, USA). Total RNA was extracted from PBMCs using the QIAamp RNA blood mini kit (Qiagen GmbH, Hilden, Germany). RNA was extracted from fat biopsies using the RNeasy mini kit (Qiagen). An Agilent 2100 Bio analyzer (Agilent Technologies, Inc., Santa Clara, CA, USA) was used to confirm the quality of extracted RNA. An ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) was used to analyse the concentration of RNA. Three hundred nanograms of RNA from each sample was reverse transcribed to complementary DNA (cDNA) using the Invitrogen superscript first strand synthesis system for real time polymerase chain reaction (RT-PCR) using the random primers (Invitrogen Corporation, Carlsbad, CA, USA). To investigate which house keeping gene to use, cDNA from PBMCs from four of the subjects from the vaccine group were analysed before and 4 h after vaccination using the TaqMan Human Endogenous Control Plate as described (Applied Biosystems, Foster City, CA, USA). Cyclophylin A was stable during inflammatory stimulation and had a suitable Ct-value. To analyse gene expression 3 μL of cDNA was mixed with TaqMan universal PCR master mix (2×) (Applied Biosystems; Branchburg, NJ, USA) and primer-probe mix (20×) with Taqman gene expression assays (Applied Biosystems) to a final volume of 25 μL. The gene expression assays used for quantitative RT-PCR (TaqMan®) were cyclophylin A (Hs99999904_m1), IL-1β (Hs00174097_m1), IL-6 (Hs00174131_m1) and TNF-α (Hs00174128_m1). Gene expression results for IL-1β, IL-6 and TNF-α were calculated as an index in relation to the cyclophylin A gene expression in each sample. The results showed that in PBMCs, there was a significant increase in TNF-α mRNA gene expression 4 h after vaccination when compared with the control group (P = 0.047). There were no differences in mRNA gene expression of IL-1β and IL-6 between the vaccinated group and controls (Fig. 1a). In AT there were no differences in mRNA gene expression of IL-1β, IL-6 or TNF-α between the vaccine and the control group (Fig. 1b). The gene expression of IL-6 was higher in AT compared with PBMC (P = 0.003) and the gene expression of IL-1β was higher in PBMC compared with AT (P = 0.0002) taking the vaccinated group and controls together. RNA expression of inflammatory cytokines in relation to house keeping genes. (a) Peripheral blood mononuclear cells (PBMC). (b) Adipose tissue. Indexes of quantitative RNA gene expression of interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumour necrosis factor-α (TNF-α) in relation to the house keeping gene cyclophylin A. Vaccinated subjects in striped boxes and controls in open boxes. Data are presented as median; box, 25–75%; whisker, nonoutlier range. Plasma levels of IL-6 were higher in the vaccine group 8 h after vaccination, 4.86 ± 9.3 pg mL−1 compared with 1.6 ± 2.0 pg mL−1 in the control group (P = 0.02). (Fig. 2). Plasma levels of interleukin-6 (pg mL−1). Vaccinated group in squares and controls in triangles. Data are presented as mean + SEM. Previous studies on the inflammatory response after vaccination have not been able to measure any increase in plasma levels of TNF-α [14, 16]. The reason for this is not clear but one can speculate that the increased TNF-α expression in PBMCs seen in the present study is due to activated monocytes/macrophages released from the vaccinated tissue. The upregulation of TNF-α gene expression might not be enough to result in detectable plasma levels of the protein but the increased plasma levels of IL-6 support the experimental model of vaccination as a stimulus to systemic inflammation. The lack of upregulation of IL-6 gene expression in PBMCs 4 h after vaccination is likely to be due to differentiation of monocytes to macrophages of the subset of PBMCs with IL-6 gene expression thereby being trapped at the site of inflammation. According to our results TNF-α is a more relevant stimulus than IL-1β in stimulating IL-6 thereby extending previous studies in animals and in vitro [4]. One previous study has shown in vivo release of IL-6 from human subcutaneous AT [6]. However, the regulation of this synthesis is largely unknown. Our results support this study by demonstrating unstimulated IL-6 gene expression in AT. Interestingly, the basal level of mRNA gene expression was fourfold higher in AT compared with PBMCs. However, we were not able to stimulate AT by vaccination. In contrast, Hoch et al. demonstrated increased transcription of IL-6 mRNA in human adipocytes stimulated ex vivo by lipopolysaccharides [17]. The reason for this discrepancy might be that we used a weaker stimulus to systemic inflammation. Furthermore, Memoli et al. showed that patients with chronic inflammation had increased IL-6 expression in AT when compared with patients with normal levels of inflammatory markers [18], suggesting that AT might respond to stimulation. Further in vivo studies with stronger stimulus to inflammation are needed to resolve this issue. Plasma levels of IL-6 in our study increased only fourfold compared with basal values. Previous results from our group have shown that plasma IL-6 levels increased 10-fold after vaccination [14]. Compared with this study, subjects did not have an indwelling peripheral vein catheter during the study, otherwise the protocol was similar. The absence of an indwelling catheter could probably explain the difference in peak plasma IL-6 levels. To avoid the risk that a haematoma caused by the fat biopsy would stimulate systemic inflammation, we compared vaccinated subjects with nonvaccinated subjects instead of using subjects as their own controls. Interestingly, the biopsy did not seem to stimulate systemic inflammation. One limitation of the present study is that fat biopsy was taken only once, at 4 h. The reason for the time chosen for fat biopsy was that we have shown that circulating IL-6 starts to increase 4 h after vaccination [14]. We cannot exclude that fat biopsy at other time-points would have resulted in a different result. In conclusion, we have shown that vaccination, a human model of inflammation, which causes endothelial dysfunction and activates coagulation [16, 19], stimulates a mild systemic inflammation but does not trigger proinflammatory gene expression in AT. Further in vivo studies including a stronger stimulus to inflammation are needed to elucidate the inflammatory capacity of AT. None. We would like to thank Camilla Hage at the Department of Cardiology, Karolinska University Hospital Solna, for excellent technical assistance. This work was supported by grants from the Swedish Heart Lung Foundation.