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    • br Materials and methods br

    2022-01-17


    Materials and methods
    Results Human adipocytes, differentiated from fibroblastic preadipocytes in culture, were incubated with IL-33 for either 3 or 24 h to examine acute and prolonged responses to the cytokine. Low, medium and high concentrations of IL-33 were employed. IL-33 had no significant effect on GPR120 mRNA level at either 3 or 24 h, except for a small, but statistically significant, reduction for the middle concentration of the cytokine at the shorter time point (Fig. 1b). There was also no effect of IL-33 on GPR41 mRNA level at 24 h; a small, dose-dependent, increase was evident at 3 h, the increase being statistically significant with the highest dose (Fig. 1c). In contrast, IL-33 induced a marked, dose-dependent increase in GPR84 mRNA at 3 h, the level with the high dose being 13.7-fold greater than in controls (Fig. 1a). This effect was, however, transitory; there was no significant change in GPR84 mRNA at 24 h (Fig. 1a). To assess the effects of IL-33 on the expression of classical inflammation-related genes, the mRNA level of a group of cytokine and chemokines was examined. IL-33 induced a marked, dose-dependent increase in mRNA of the ILB, IL6, CCL2, CXCL2 and CSF3 genes at 3 h (Fig. 2a–e). With the highest dose of IL-33, there were 3.0-, 11.7-, 15.2- and 15.6-fold increases in mRNA level of the CCL2, IL6, CXCL2 and ILB genes, respectively. CSF3 exhibited a very substantial response to IL-33, the mRNA level with the highest dose of the cytokine being 183-fold higher than in the controls (Fig. 2e). As with GPR84, these responses to IL-33 were transitory; there was no effect of IL-33 on IL-1β mRNA at 24 h (Fig. 2a), and there was actually a small reduction in CCL2 mRNA with the low and medium doses of the cytokine (Fig. 2c). CSF3, IL-6 and CXCL2 mRNA levels were each elevated at 24 h with the highest dose of IL-33 (3.9-, 2.5- and 3.3-fold, respectively); however, for CSF3, the increase was substantially lower than the 183-fold increase at 3 h. In Thirty percent of to the other adipokine genes, expression of ADIPOQ, which encodes the key adipocyte hormone adiponectin, was not modified by IL-33, the mRNA level being unchanged at both 3 and 24 h with each dose of the cytokine (Fig. 2f). IL18 expression was also unaltered (results not shown). To determine whether the changes in cytokine and chemokine gene expression were reflected by similar changes in secretion of the encoded protein, the effect of IL-33 on the release of IL-6 and G-CSF was examined. Treatment with IL-33 resulted in a dose-dependent increase in the amount of the two adipokines in the culture medium at both 3 and 24 h (Fig. 2g and h). The amount of both proteins was greater at 24 h than at 3 h, reflecting the delay between increases in mRNA level and release of the protein product. At 24 h, with the highest dose of IL-33, the amount of IL-6 was 7.9-fold greater than in the controls (4.7-fold at 3 h) and 23.9-fold higher (4.6-fold at 3 h) in the case of G-CSF.
    Discussion This study demonstrates that expression of the GPR84 fatty acid receptor/sensor gene in human adipocytes is strongly and acutely stimulated by IL-33. This parallels the stimulatory effect of IL-1β and TNFα, as well as other mediators, on the expression of this gene in fat cells [4], [7], [8], [9]. GPR84 is considered a pro-inflammatory receptor, its activation amplifying the production of IL-8 and IL-12 p40 in polymorphonuclear leukocytes and of TNFα by macrophages [5]. Thus IL-33 may contribute to the inflammatory response in adipose tissue in part through increased production of GPR84. IL-33 did not have a suppressive effect on GPR120 expression, in contrast to TNFα and IL-1β [7], [8]. The insulin sensitising and anti-inflammatory actions of GPR120, which is a receptor for n-3 polyunsaturated fatty acids [2], [3], is unlikely to be modified by IL-33 contrary to what has been projected with IL-1β and TNFα [7]. The intracellular signalling pathways linked to the IL-33 receptor, ST2 (and its co-receptor IL-1 receptor accessory protein) [15], which are expressed in human adipocytes [11], would appear to be implicated in the modulation of the transcription of the GPR84 gene, but not GPR120. It is noted that while 3 and 24 h time-points were employed, reflecting potential acute and sustained effects of IL-33, the possibility cannot be excluded that some changes might occur at intermediate times, but if so these are likely to be limited.