Zinc transporter SLC39A7 relieves zinc deficiency to suppress alternative macrophage activation and impairment of phagocytosis

Abstract

Macrophages are key phagocytic cells and play an important role in eliminating external microorganisms and endogenous danger signals. Dysregulation in macrophage functions have been reported in patients with asthma. Zinc homeostasis is critical in maintaining macrophage functions. The solute carrier (SLC) protein SLC39A7, a Zn2+ importer, has recently been linked to asthma. However, the roles of SLC39A7 in macrophage phagocytosis are not well understood. Here we found that phagocytosis efficiency was significantly decreased in SLC39A7-knockdown THP-1 cells, however the phagocytosis capability could be reversed with zinc supplementation. SLC39A7 deficiency skewed macrophages towards alternative activation, as indicated by increased expression of M2 activation marker CD206 and decreased expression of M1 activation marker NOS2. Consistent to this result, SLC39A7-knockdown cells produced reduced amounts of proinflammatory cytokines TNF- and IL-6. Furthermore, the mRNA level of receptor Clec4e previously known to be involved in phagocytosis of BCG was significantly reduced in SLC39A7 knockdown cells. Importantly, all these defects due to SLC39A7 deficiency could be reversed by zinc supplementation. Thus, zinc transporter SLC39A7 provide support for phagocytosis and classical macrophage activation.

Fig 1. SLC39A7 was up-regulated in macrophages in response to BCG-p stimulation.

The mRNA (A) and protein (B) of SLC39A7 was measured at 0 h, 6 h, 24 h after infection with BCG-p (MOI 5:1) in THP-1 cells; the experiment was performed three times. (A) was analyzed by one-way ANOVA, **p < 0.005.

Fig 2. Knockdown of SLC39A7 reduced the proliferation of THP-1 cells.

(A) Western blot of SLC39A7 knockdown cell lines. Equal amount of THP-1 control and SLC39A7 knockdown cell lysates were probed with anti-SLC39A7 antibody (Proteintech). (B) Proliferation of two SLC39A7 knockdown cell lines was quantitated by CCK8 assay. The result was analyzed by two way ANOVA, ***p < 0.001 compared to control (NC). (C) The percentage of living cells was determined from SYTOX Green staining under fluorescence microscopy. The cells was stained by 10 nM SYTOX Green at 72 h after PMA stimulation for 15min, then fixed by 4% paraformaldehyde, the nucleus were stained by DAPI before observing using fluorescence microscopy. (D) The cell adherence rates of SLC39A7 knockdown cells and control (NC) following stimulation with 100nM PMA for 24, 48 and 72h. SLC39A7-knockdown macrophages were treated with 5 μM ZnCl2 and 0.5 μM pyrithione. Experiments were repeated three times with similar observations, and representative data was shown. The data was analyzed by two-way ANOVA, *P < 0.05, **p < 0.005, ***p < 0.001, ****P < 0.0005, ns: not significant.

Fig 3. The efficiency of phagocytosis to BCG-GFP in SLC39A7 KD THP-1 cells.

(A) Images from GFP-expressing BCG-p (green) infected THP-1 macrophages, the nuclei (blue) were stained using DAPI. (B) Phagocytosis of GFP-expressing BCG-p in SLC39A7 knockdown cells or control (NC). GFP-expressing BCG infected SLC39A7 KDs or NC were measured by flow cytometry. (C) Colony-forming units (CFU) of BCG-p at 3 h. (B) and (C) were analyzed by one-way ANOVA, **P < 0.005, ***p < 0.001, ****P < 0.0005.

Fig 4. The mRNA expression of M1/M2 markers in SLC39A7 KD THP-1 cells.

The mRNA Level of Arg-1, Ym-1, CD206 (A), NOS2, CD11C (B) in knockdown cells or control response to BCG-p infection at 6 h (MOI = 10). Data was representative of three independent experiments and expressed as mean ± SD for three biological replicates. All data was analyzed by two-way ANOVA, *P < 0.05, **P < 0.005, ***p < 0.001, ****P < 0.0005, ns: not significant.

Fig 5. The cytokines expression in SLC39A7 KD THP-1 cells.

the mRNA levels of TNF-α, IL-6 and IL-10 were tested in knockdown and control cells at 6 h after BCG-p infection (A), and the concentrations of TNF-α and IL-6 in the supernatants were examined at 48h after BCG-p infection by ELISA kits according the manufacturer's protocol (NeoBioscience) (B). The data was analyzed by two-way ANOVA, *P < 0.05, **P < 0.005, ***p < 0.001, ****P < 0.0005, NS: not significant.

Fig 6. The mRNA expression of cell surface receptors in SLC39A7 KD THP-1 cells.

The mRNA of Clec4e, TLR4, DC-SIGN, MARCO, Dectin-1, Clec4d in knockdown cell or control response to BCG-p infection at 6 h (MOI = 10). All data was analyzed by two-way ANOVA, *P < 0.05, **P < 0.005, ***p < 0.001, ****P < 0.0005, NS: not significant.