Loss of FADS2 function severely impairs the use of HeLa cells as an in vitro model for host response studies involving fatty acid effects.

Established epithelial cell lines equipped with pattern recognition receptors such as the Toll-like receptor (TLR)-2 are common tools for immune response studies on invading pathogens, e.g. the obligate intracellular species of Chlamydia. Moreover, such models are widely used to elucidate fatty acid-mediated immune effects. In several transformed cell lines, however, unusual loss of metabolic functions was described. The cell lines A549 and HeLa are poorly characterized in this respect. Therefore, we comparatively assessed the metabolic capacity of A549 and HeLa prior to proposed application as in vitro model for fatty acid effects on chlamydial infection.We incubated both cell lines either with substrates (C18:2n-6 or C18:3n-3) or products (C18:3n-6, C18:4n-3) of fatty acid desaturase-2 (FADS2), and analysed the fatty acid profiles after 24 h and 72 h by gas chromatography. Based on these data, we suspected that the complete discontinuation of normal biosynthesis of long-chain polyunsaturated fatty acids (LC-PUFA) in HeLa was due to loss of FADS2 function. Consequently, prostaglandin E2 (PGE2) formation was less inducible by TLR2 stimulation in HeLa, likely as a result of not only insufficient supply of precursors but also weak cyclooxygenase-2 (COX-2) response. In accordance, Chlamydia infection rates were consistently lower in HeLa than in A549. Sequence analysis revealed no alteration within the FADS2 gene in HeLa. The FADS2 expression level, however, was significantly lower and, in contrast to A549, not regulated by C18:2n-6. A549 exhibited regular fatty acid metabolism and enzyme functionality.Our data show that HeLa cells considerably differ from A549 at several stages of fatty acid metabolism. The poor metabolic potential of HeLa, mainly concerning FADS2 upstream of COX-2 function, calls into question whether these cells represent a good model to unveil fatty acid or downstream eicosanoid effects in the course of intracellular bacterial infection

Primers used for <i>FADS2</i> encoding sequence analysis.

<p>tr. var. - transcript variants.</p><p>Primers used for <i>FADS2</i> encoding sequence analysis.</p

Primers and UPL probes used for real-time gene expression analysis.

<p>*Products on intended targets: transcript variants 1–3.</p><p>Primers and UPL probes used for real-time gene expression analysis.</p

Partial chromatograms of cellular lipids of HeLa and A549.

<p>Cells were incubated without (DMSO-treated control) or with the indicated fatty acid (33 µM) for (<b>A</b>) 24 h or (<b>B</b>) for 72 h. 1–C18∶0, 2–C18∶1<i>c</i>9, 3–C18∶1<i>c</i>11, 4–C18∶2<i>n</i>−6, 5–C18∶3<i>n</i>−6, 6–C20∶2<i>n</i>−6, 7–C20∶3<i>n</i>−6, 8–C20∶4<i>n</i>−6, 9–C22∶4<i>n</i>−6, 10–C22∶5<i>n</i>−6, 11–C18∶3<i>n</i>−3, 12–C20∶5<i>n</i>−3, 13–C22∶5<i>n</i>−3, 14–C22∶6<i>n</i>−3. <b>A:</b> C20∶2<i>n</i>−6 (peak 6) was the only metabolite identified in HeLa following incubation with C18∶2<i>n</i>−6. In contrast, all detectable elongated and desaturated products of C18∶2<i>n</i>−6 increased in A549 (filled-headed arrows). When C18∶3<i>n</i>−6 was the supplement, the respective metabolites increased in HeLa, too (broken-lined arrows). <b>B:</b> As a product of C18∶3<i>n</i>−3, C22∶6<i>n</i>−3 (peak 14) increased in A549 after 72 h.</p

Real-time quantitative expression of <i>FADS2</i> mRNA in HeLa and A549.

<p>Cells were incubated without (DMSO-treated control) or with 33 µM C18∶2<i>n</i>−6 or C18∶3<i>n</i>−6 for 24 h. Different letters indicate significant differences: a <i>vs.</i> b, and b <i>vs.</i> c, p≤.005.</p

Metabolic capacity of HeLa compared to A549.

<p>Fatty acid profiles were determined by GC-FID analysis of cellular lipid extracts and are expressed as % of total FAME. Data represent means ± SD.</p>a<p>Cells were incubated without (−) or with (+) 33 µM of C18∶2<i>n</i>−6 and C18∶3<i>n</i>−3, respectively, for 24 h. Both are substrates for FADS2.</p>b<p>Cells were incubated without (−) or with (+) 33 µM of the FADS2 products C18∶3<i>n</i>−6 and C18∶4<i>n</i>−3 for 24 h.</p>c<p>Considered Δ8-desaturation products.</p>d<p>Synthesis requires upstream a second step of <i>FADS2</i>-encoded activity.</p>e<p>After 72 h, significant increase in A459 to 2.7±0.2 when incubated with C18∶3<i>n</i>−3 (FA treatment×cell line: p = .002; see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115610#pone-0115610-g001" target="_blank">Figure 1B</a>, peak 14) and to 2.8±0.1 when incubated with C18∶4<i>n</i>−3 (FA treatment×cell line: p = .011).</p><p>Metabolic capacity of HeLa compared to A549.</p