Downregulation of SLC7A7 Triggers an Inflammatory Phenotype in Human Macrophages and Airway Epithelial Cells

Abstract

Lysinuric protein intolerance (LPI) is a recessively inherited aminoaciduria caused by mutations of SLC7A7, the gene encoding y+LAT1 light chain of system y⁺L for cationic amino acid transport. The pathogenesis of LPI is still unknown. In this study, we have utilized a gene silencing approach in macrophages and airway epithelial cells to investigate whether complications affecting lung and immune system are directly ascribable to the lack of SLC7A7 or, rather, mediated by an abnormal accumulation of arginine in mutated cells. When SLC7A7/y+LAT1 was silenced in human THP-1 macrophages and A549 airway epithelial cells by means of short interference RNA (siRNA), a significant induction of the expression and release of the inflammatory mediators IL1β and TNFα was observed, no matter the intracellular arginine availability. This effect was mainly regulated at transcriptional level through the activation of NFκB signaling pathway. Moreover, since respiratory epithelial cells are the important sources of chemokines in response to pro-inflammatory stimuli, the effect of IL1β has been addressed on SLC7A7 silenced A549 cells. Results obtained indicated that the downregulation of SLC7A7/y+LAT1 markedly strengthened the stimulatory effect of the cytokine on CCL5/RANTES expression and release without affecting the levels of CXCL8/IL8. Consistently, also the conditioned medium of silenced THP-1 macrophages activated airway epithelial cells in terms of CCL5/RANTES expression due to the presence of elevated amount of proinflammatory cytokines. In conclusion, our results point to a novel thus far unknown function of SLC7A7/y+LAT1, that, under physiological conditions, besides transporting arginine, may act as a brake to restrain inflammation.

Keywords: LPI; arginine; cytokine and chemokines; inflammation; innate immunity.

Figure 1

THP-1 cells, routinely grown in the presence of 1 mM extracellular arginine, were transfected with scrambled or SLC7A7 short interference RNA (siRNA) in the presence of 80 nM phorbol-12-myristate-13-acetate (PMA), as described in Section “Materials and Methods”; after 96 h, cells were analyzed for the expression of SLC7A7 mRNA with RT-qpolymerase chain reaction (RT-qPCR) (A) and for intracellular arginine content by means of HPLC/ESI-MS-MS (B). In the same cells, the production and release of IL1β and TNFα were monitored with RT-qPCR (C) and ELISA assay (D), respectively. Data are mean ± SEM of four different determinations, each performed in duplicate. *p < 0.05, **p < 0.01, ***p < 0.001 vs. scrambled siRNA with One sample t-test (A,C) or Student’s t-test for paired samples (B,D).

Figure 2

THP-1 cells were grown in the presence of the indicated concentrations of extracellular arginine. (A–C) Cells were differentiated to macrophages by exposure to 80 nM phorbol-12-myristate-13-acetate (PMA) for 96 h, then arginine content was measured by means of HPLC/ESI-MS-MS (A), while the expression of IL1β (B) and TNFα (C) mRNAs was monitored with RT-qpolymerase chain reaction (RT-qPCR). Data are mean ± SEM of five independent determinations. *** p < 0.001 vs. [Arg]_{extracellular} 0.1 mM. (D–F) Cells were transfected with scrambled or SLC7A7 short interference RNA (siRNA) along the PMA-induced differentiation, as described in Section “Materials and Methods,” and analyzed for the expression of SLC7A7 (D), IL1β (E), and TNFα (F) mRNAs with RT-qPCR. Data are mean ± SEM of four different determinations, each performed in duplicate. *p < 0.05, **p < 0.01, ***p < 0.001 vs. scrambled siRNA with one-way ANOVA followed by Bonferroni post hoc test.

Figure 3

THP-1 cells, routinely grown in the presence of 1 mM extracellular arginine, were transfected for 96 h with scrambled or SLC7A7 short interference RNA (siRNA) during PMA-induced differentiation, as described in Section “Materials and Methods”; when indicated, gene silencing was performed in the absence (none) or in the presence of caspase-1 specific inhibitor (z-WHED-FMK). (A–C) The expression of IL1B mRNA was determined with RT-RT-qPCR (A), IL1β release was measured with ELISA assay (B), and the activity of caspase-1 was determined as described in Section “Materials and Methods” (C). Data are mean ± SEM of three different determinations, each performed in duplicate. *p < 0.05, **p < 0.01, ***p < 0.001 vs. scrambled siRNA; ^$$p < 0.01 vs. SLC7A7 siRNA with one-way ANOVA followed by Bonferroni post hoc test. (D) The amount of NFκB-p65 in the nuclear fraction was monitored with Western Blot analysis. The experiment was repeated twice with comparative results; a representative blot is shown.

Figure 4

(A) A549 cells were washed in Earle’s balanced salt solution (EBSS) (Na⁺-present) or Na⁺-free NMG-EBSS (Na⁺-absent), as indicated; arginine uptake was then assayed with 1-min incubation in the same solution supplemented with l-[³H]arginine (0.05 mM; 3 μCi/ml) in the absence (total) or in the presence of 2 mM leucine or 2 mM leucine + lysine. Data are mean ± SD of five independent determinations in a representative experiment, which, repeated twice, gave comparable results. ***p < 0.001 vs. total uptake with one-way ANOVA followed by Bonferroni post hoc test. (B) Data shown in Panel (A) were employed to calculate the relative contribution of systems y⁺L and y⁺ to total arginine uptake (see Materials and Methods and Results).

Figure 5

A549 cells were transfected with scrambled, SLC7A6 or SLC7A7 short interference RNA (siRNA), as described in Section “Materials and Methods.” The expression of SLC7A6 (A), SLC7A7 (B), and IL1B and TNFA (F) mRNA was measured with RT-qpolymerase chain reaction (RT-qPCR); data are mean ± SEM of three independent determinations, each performed in duplicate. Protein levels of y+LAT2 and y+LAT1 were determined with Western Blot analysis (C); a representative blot is shown. The transport activity of System y⁺L (D) and arginine intracellular content (E) were also measured, as described in Section “Materials and Methods”; data are mean ± SEM of four independent experiments, each performed in quadruplicate. *p < 0.05, **p < 0.01, ***p < 0.001 vs. scrambled siRNA with one-way ANOVA followed by Bonferroni post hoc test.

Figure 6

(A,B) A549 cells were incubated in the presence of the indicated concentrations of IL1β; after 24 h, the expression of CXCL8/IL8 and CCL5/RANTES was monitored with RT-qpolymerase chain reaction (RT-qPCR). Representative experiments are shown. (C,D) A549 cells were transfected with scrambled or SLC7A7 short interference RNA (siRNA) for 96 h (see Materials and Methods); where indicated, IL1β (10 ng/ml) was added to the incubation medium for the last 24 h; at the end, the expression and release of the indicated chemokines was monitored at mRNA (C) and protein (D) level with RT-qPCR and ELISA, respectively. Data are mean ± SEM of three independent determinations, each performed in duplicate. *p < 0.05, **p < 0.01 vs. scrambled siRNA; ^$p < 0.05 vs. scrambled siRNA + IL1β with one-way ANOVA followed by Bonferroni post hoc test.

Figure 7

A549 were incubated with fresh medium (left panels) in the presence of caspase-1 inhibitor (+ z-WHED-FMK); in parallel, another set of A549 cells (right panels) was incubated with conditioned medium (CM) of THP-1 cells (CM-THP-1) silenced for 96 h with scrambled or SLC7A7 short interference RNA (siRNA), either in the absence or in the presence of caspase-1 inhibitor. After 24 h, the expression of CXCL8/IL8 and CCL5/RANTES was monitored with RT-qpolymerase chain reaction (RT-qPCR). Data are mean ± SEM of three independent determinations, each performed in duplicate. **p < 0.01 vs. scrambled siRNA CM-THP-1; ^$p < 0.05 vs. SLC7A7 siRNA CM-THP-1 with one-way ANOVA followed by Bonferroni post hoc test.

Figure 8

Schematic representation of the pathogenic model proposed for pulmonary complications in LPI.