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

Wenyan Xie; Qinghua Xue; Liangfei Niu; Ka-Wing Wong

doi:10.1371/journal.pone.0235776

. 2020 Jul 9;15(7):e0235776. doi: 10.1371/journal.pone.0235776

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

Wenyan Xie ^1,^#, Qinghua Xue ^1,^#, Liangfei Niu ¹, Ka-Wing Wong ^1,^*

Editor: Rebecca A Hall²

PMCID: PMC7347223 PMID: 32645059

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.

Introduction

Macrophages are vital first-line cells in host defense. To accomplish this task macrophages possess three critical functions: phagocytosis, antigen presentation, and immune regulation. They are essential for maintaining a normal immune state under various pathophysiological conditions [1]. Zinc homeostasis was found to be important for the innate immune system, especially for maintaining the function of macrophages [2]. Zinc plays an important role in monocyte adhesion, migration and differentiation into tissue macrophages [3], and phagocytosis of macrophages and its cytokine production [4]. Zinc supplementation increases the phagocytosis of E. coli and Staphylococcus aureus by peritoneal macrophages in a mouse model of polymicrobial sepsis [5], and inhibits alternative macrophage polarization [6]. Insufficient zinc levels in cells can lead to excessive inflammatory response, producing excess cytokines and chemokines such as TNF-α, IL-6, IL-8 and IL-10 [7].

Homeostasis of intracellular zinc mainly depends on two families of zinc transporters: SLC39A/ZIP and SLC30A/ZnT. The SLC39A family members facilitate zinc influx to cytoplasm from extracellular spaces as well as intracellular compartments such as endoplasmic reticulum (ER) [8], whereas the SLC30A family members transport zinc in the opposite direction away from cytoplasm [9]. SLC39A7 is the only known ZIP member that is localized to the ER membrane [10] and is essential for regulation of cytosolic zinc levels [11]. Deletion of the SLC39A7 gene in mesenchymal stem cells leads to the accumulation of zinc in the ER, triggering overexpression of the UPR gene and endoplasmic reticulum stress [12]. Recent data shows that SLC39A7 is implicated in glucose metabolism and glycemic control in skeletal muscle cells [13, 14]. For immune cells, SLC39A7 is essential for B cell development and BCR signaling [15]. Although SLC39A7 is shown to regulate the immune system, the effect of SLC39A7 on the phagocytosis and macrophage activation is poorly understood. A transcriptional profile study identified SLC39A7 as gene product associated with asthma [16]. Asthma is associated with impaired macrophage phagocytosis, alternative macrophage differentiation and zinc deficiency. BCG is a vaccine to prevent tuberculosis and has also been used as a potent immunomodulator in the decades [17]. BCG has been reported that the protection against asthma [18]. According to these reports, we hypothesized SLC39A7 played a critical role in macrophage phagocytosis of BCG. We generated SLC39A7-knockdown THP-1 cell lines by using CRISPR-Cas9 gene editing system. Our results revealed phagocytosis impairment and alternative macrophage polarization in SLC39A7-knockdown THP-1 cells. And importantly, these defects could be rescued with Zn2+ supplementation.

Results

1. Expression of SLC39A7 increased in BCG infected macrophages

We first determined whether expression of SLC39A7 was regulated in macrophages during infection of BCG Pasteur stain (BCG-p). THP-1 cells were differentiated into macrophages by PMA, and then infected with BCG at a multiple of 5 (MOI). Western blotting and quantitative PCR were performed to detect the expression of SLC39A7 after infection at 6h and 24h. Both of mRNA and protein level of SLC39A7 were significantly increased in BCG-p infected THP-1 cells compared with uninfected macrophages (Fig 1A and 1B and S1 Fig).

Fig 1 — 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.

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

To evaluate the function of SLC39A7 during infection in macrophages, we used the CRISPR-Cas9 gene editing system to generate SLC39A7-knockdown cell lines. Western blot with anti-SLC39A7 antibody showed that expression of SLC39A7 protein was abolished in SLC39A7-knockdown cell lines (Fig 2A and S2 Fig). Cell proliferation after 4d as measured by CCK8 assay was significantly lower in SLC39A7-knockdown cells (KD-2 and KD-4) than in non-target transfected control cells (NC) (Fig 2B). Adherence rate of the SLC39A7-knockdown cells was lower than that of NC after PMA stimulation (Fig 2D). Furthermore, we determined whether supplementation of exogenous Zn²⁺ could reverse the adherence defect. The result showed that the addition of ZnCl₂ and pyrithione, an ionophore that transports zinc through cell membrane, rescued the adhesion defect at 72 h after PMA stimulation (Fig 2D). Cell survival rates, measured at 72 h after PMA stimulation by nucleic acid stain SYTOX green that only stained DNA in dead cells, were indistinguishable between knockdown and control cells (Fig 2C). Thus, SLC39A7 knockdown reduced the rate of proliferation and adhesion by PMA-stimulated THP-1 cells.

Fig 2 — (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.

3. Knockdown of SLC39A7 disrupted phagocytosis of BCG-p by THP-1 cells

Phagocytosis to pathogens by macrophages is a key innate immune effector mechanism. To evaluate the role of SLC39A7 in the host response to BCG-p infection we examined the phagocytic capacity of SLC39A7 knockdown cells and non-targeting control (NC). Differentiated THP-1 were infected with GFP-expressing BCG-p for 4 h at a multiplicity of infection (MOI) of 5. We analysis the phagocytosis to BCG-p in THP-1 cells through fluorescence microscopy, flow cytometry, and colony-forming units (CFU) (Fig 3). Compared with NC, phagocytosis efficiency by SLC39A7-knockdown THP-1 cells was dramatically decreased (Fig 3A and 3B). CFUs in SLC39A7-knockdown cells were also lowered relative to the NC cells (Fig 3C). These results showed that the phagocytosis capacity to BCG-p was significantly reduced in SLC39A7-knockdown THP-1 cells.

Fig 3 — (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.

Since SLC39A7 knockdown is known to reduce intracellular concentration of Zn²⁺ [11] we examined intracellular Zn2+ concentration using Fluozin-3^TM AM and found that, as shown in previous report, the intracellular Zn²⁺ level in SLC39A7 knockdown cells was lower in SLC39A7-knockdown cells than in control cells (S3 Fig). To determine whether Zn2+ supplement could rescue the phagocytosis defect of BCG-p by the SLC39A7-knockdown THP-1 cells we found that supplementation of Zn2+ ionophore pyrithione (0.5 μM) and ZnCl₂ (5 μM) significantly improved the phagocytic capacity of SLC39A7-knockdown cells (Fig 3B). This result was confirmed by measuring the level of intracellular viable BCG-p (Fig 3C). Our results therefore indicated that SLC39A7 could play a role in the macrophages phagocytosis.

4. The mRNA expression of markers and cytokines associated with M1/M2 macrophage activation in SLC39A7-knockdown THP-1 cells

To study the role of SLC39A7 in macrophage phagocytosis we analyzed the mRNA expression of macrophage genes associated with classical M1 activation (NOS2 and CD11c) and alternative M2 activation (Arg-1, Ym-1, and CD206). mRNA expression of M2 macrophage genes Arg-1 and Ym-1 and M1 genes CD11c was either unchanged in knockdown cells or unresponsive to zinc supplementation (Fig 4A and 4B). However, expression of M2 macrophage gene marker CD206 was up-regulated in one of the SLC39A7-knockdown cells (KD4) and this up-regulation could be reversed by zinc supplementation (Fig 4A). Expression of M1 marker NOS2 was down-regulated in SLC39A7-knockdown cells and this down-regulation was reversible by zinc supplementation (Fig 4B).

Fig 4 — 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.

Classically activated M1 macrophage has a proinflammatory profile dominated by cytokines TNF-α and IL-6, whereas alternatively activated M2 macrophage has an anti-inflammatory profile dominated by IL-10. Although no evidences supporting a role for SLC39A7 in regulating expressions of TNF-α, IL-6, and IL-10 in a fashion reversible by zinc supplementation were found (Fig 5A), secretion of TNF-α and IL-6 proteins at 48h after BCG-p infection were reduced in SLC39A7-knockdown cells than in control cell and this reduction in secretion of TNF-α and IL-6 was reversible by zinc supplementation (Fig 5B). IL-10 protein was undetectable at 48h after BCG-p infection. Taken together, our results indicate that SLC39A7 likely play a role in classical M1 activation of macrophages.

Fig 5 — 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.

5. The expression of Clec4e was reduced in SLC39A7-knockdown cells

Bacteria use their cell surface associated macromolecular structures to interact with specific receptor molecules on host cells. We therefore examined the mRNA expression of cell surface receptors previously known to be involved in phagocytosis. The results showed that the mRNA level of receptors Clec4e (also known as Mincle) was significantly decreased and TLR4 mRNA level was increased in SLC39A7 knockdown cells (Fig 6). However, Clec4e expression increased significantly and TLR4 expression decreased when exogenous Zn²⁺ was added in the knockdown cells. In contrast, the mRNA levels of other surface receptors DC-SIGN, MARCO, Dectin-1 and Clec4d were comparable with that in control. The deficiency of SLC39A7 could reduce the transcription level of Clec4e.

Fig 6 — 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.

Discussion

Macrophages are critical first responders. They are specialized to recognize and eliminate pathogens [19]. The level of intracellular zinc is known to influence the phagocytosis capacity of macrophages [20]. SLC39A7, the only known SLC39A family member localized on the ER membrane [10], is essential for regulation of cytosolic zinc level [11]. Previous studies have examined the role of SLC39A7 on Notch [21] and insulin signaling [22–24]. However, the role of SLC39A7 in phagocytosis has not been previously examined. In this report, we generated SLC39A7 knockdown THP-1 cell lines by using CRISPR-Cas9 gene editing system. Our results showed that phagocytosis was severely inhibited in the SLC39A7 knockdown THP-1 cells. Importantly, this defect in phagocytosis could be restored by supplementation of exogenous Zn²⁺. Thus, SLC39A7 plays a role in the phagocytosis of BCG by THP-1 cells.

Macrophages possess a broad array of cell surface receptors, intracellular mediators and essential secretory molecules for recognition, engulfment and destruction of invading pathogens and also for regulation of other kinds of immune cells [25]. The C-type lectin receptor Clec4e plays a role in eliciting inflammatory response against mycobacterium and mediates neutrophil phagocytosis and extracellular trap formation [26]. We found that the expression of Clec4e was dramatically decreased in SLC39A7 knockdown cells. This result could explain our observation that SLC39A7 knockdown negatively affected phagocytosis.

Toll-like receptors (TLRs) play a crucial function in macrophage maturation and activation. TLR4 expressed by macrophages are among the first line of defense against pathogens [27]. The expression of TLR4 was slightly increased in SLC39A7 knockdown cells. This effect could be due to increased cell death in SLC39A7 knockdown cells. The deficiency of SLC39A7 is known to cause cell death and release of the extracellular inflammatory factor HMGB1, which could activate TLR4 expression [28]. Moreover, TLR4 activation impaired phagocytic capacity of microglia [29]. It could further explain the defective phagocytosis in SLC39A7 knockdown cells.

Macrophages are highly plastic and can be differentiated into two major phenotypic subsets in response to different ambient stimulus: pro-inflammatory M1 phenotype and anti-inflammatory M2 phenotype [30]. Cellular zinc homeostasis could modulate differentiation of macrophages [31]. It is known that high Zn2+ promotes M1 differentiation while inhibits M2 differentiation, while Zn2+ deficiency inhibits the differentiation of M1. Given that SLC39A7 deficiency lowered the cytosolic Zn2+ levels, we investigated the role of SLC39A7 in the differentiation of macrophages and measured gene expression of M1/M2-associated markers and cytokines. In accordance with previous studies [32–34], we selected CD11c and NOS2 as markers of M1 macrophage activation, and Arg-1, Ym-1 and CD206 as indicators of M2 macrophage responses. Compared with wild type cells, the mRNA of the M2 marker CD206 increased significantly in KD4 cells, and the expression of M1 marker NOS2 was decreased in both KD2 and KD4 cells. Moreover the pro-inflammatory cytokines TNF-α, which induce macrophage activation and cytotoxic activity [35], was decreased in SLC39A7 knockdown cells. IL-10 is a well-known anti-inflammatory expression increased and IL-10 plays a negative role in BCG-induced macrophage cytotoxicity [36]. One of the KD cells KD-2 did not exhibit defects in several cytokine expressions (Fig 4A and 4B), and was unresponsive zinc supplementation. We presumed that the different adhesion of the two knockdown cells caused the discrepancy. KD-4 has a bigger defect in adhesion and a more responsiveness in cytokine productions. It is interesting to note that monocytes from systemic lupus erythematosus have defects in adhesion and produce elevated levels of IL-6, TNF-a, and IL-10 than healthy monocytes [37]. Thus, the discrepancy between KD-2 and KD-4 could reflect a differential compensatory response by the KD cells. In summary, our results indicated that SLC39A7 could regulate macrophage polarization and balance of pro-inflammatory and anti-inflammatory responses.

Our results might have significances towards the pathophysiology of asthma in humans. Alternative M2 macrophage activation and impaired macrophage phagocytosis were two characteristics in patients with asthma [38, 39]. Interestingly, patients with asthma are known to have significantly lower plasma levels of Zn²⁺ [40]. It is possible that this Zn²⁺ deficiency is responsible for the M2 macrophage activation, and impaired macrophage phagocytosis. To compensate for the Zn²⁺ deficiency, SLC39A7 is upregulated in patients with asthma than healthy controls as previously reported [16]. Recent data shows that SLC39A7 is implicated in glucose metabolism and glycemic control in skeletal muscle cells [13, 14]. SLC39A7 has essential role in B cell development, SLC39A7 was required for BCR signaling [15]. All these observations suggested that SLC39A7 played an essential role in the host immune metabolism. The function of SLC39A7 should be further investigated.

Materials and methods

Cell culture

The human myelogenous leukemia cell line THP-1 was purchased from ATCC (Manassas, VA). The cells were maintained in RPMI 1640 with 10% fetal bovine serum at 37˚C in a 5% CO2 humidified incubator.

Generation of Knockdown cell line with CRISPR/Cas9

Guide RNA sequences for CRISPR/Cas9 were designed at CRISPR design web site (http://crispr.mit.edu/), provided by the Feng Zhang Lab. The SLC39A7 gRNA sequences were shown as follows: gRNA2:5’-CACCGCTCTCCCTCACCAGGCACTG-3’and gRNA4: 5’-CACCGAGCTGCTGAGATCAGCACTG-3’. The complementary oligonucleotides for gRNAs were annealed, and cloned into LentiCRISPRv2 (Addgene, Cambridge, MA). THP-1 cells were transfected with LentiCRISPRv2/gRNA using Lipofectamine 2000 (ThermoFisher, Waltham, MA, USA) following the manufacturer’s recommended protocol. Two days after transfection, cells were treated with 1 μg/ml of puromycin (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) for three days. After two weeks, the single cell was isolated with Flow cytometry into 96 well plates. The knockdown efficiency was evaluated with the protein expression of SLC39A7 by western blot.

Cell Counting Kit-8 (CCK8) assay

The cell proliferation potential of SLC39A7 knockdown was measured with CCK-8 assay (Dojindo) according to the manufacturer’s instruction. In brief, the indicated cells (at a cell density of 1.5×104 cells /well) were seeded into a 96-well plate and cultured in normal growth medium. 10 μl CCK-8 working solution was added to each well and incubated at 37°C for 4 h. The absorption at 450 nm was measured at indicated time points (24, 48, 72, and 96 h, respectively) using an ELISA plate reader. Experiments were performed in triplicate.

Cell adherence assay

THP-1 cells (3x106 cells/well) were seeded in 24-well plates with 100nM phorbol myristate acetate (PMA) (Sigma-Aldrich) for 0, 1, 2 and 3 days, respectively. Unattached cells in the supernatant were counted. The cell adherence rate was calculated according to the cell number of THP-1 cells without stimulation.

Bacterial strains and cultures

The Mtb strain BCG-Pasteur (BCG-p) or BCG-p harboring the plasmid pMF42A encoding green fluorescent protein (GFP) were grown on Middlebrook 7H9 medium (Difco, Becton Dickinson, Franklin Lakes, NJ, USA) with complete supplements [10% OADC, 0.5% glycerol and 0.05% Tween-80]. BCG-p was grown to logarithmic phase or an OD600 of between 0.45 and 0.85. Before infection, bacteria were pelleted in a 2ml tube with attached loop cap, suspended in 1 ml of PBS in a 15 ml conical tube, and were sonicated three time for 15 s (80 output and 100% duty cycle). The OD600 of each strain was then measured. 1 OD600 is equal to about 3×108 CFU ml-1. For infection of THP-1, 10% normal human serum (Gemini, CA, USA) was used to facilitate BCG-p binding to host cells. After 3 h incubation at 37°C, infected cells were washed three times gently with room temperature PBS and then maintained in RPMI 1640 containing 10% fetal bovine serum (FBS).

Reverse transcription and quantitative real-time PCR

Total RNA was extracted using Trizol reagent (Takara), according to the manufacturer’s protocol. The fist-stand cDNA was synthesized using ImProm-II Reverse Transcription System (Promega) according to the manufacturer’s instructions. Quantitative PCR was performed on the Applied Biosystems 7500 Real-Time PCR System (ABI, USA) with SYBR Premix Ex Taq (Takara). Expression of each gene was normalized to the expression of the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Data was analyzed using the 2-ΔΔCt method. PCR for each sample was performed in triplicates. The sequences of primers are provided in the S1 Table.

ELISA

The supernatants were collected at 48h after BCG-p infection. The level of TNF-α and IL-6 was determined by ELISA using ELISA kits according the manufacturer's protocol (NeoBioscience).

Phagocytosis assay

Differentiated macrophages were infected with GFP-expressing BCG-p for 4 h at a multiplicity of infection (MOI) of 5. After the infection, cells were washed three times gently with room temperature PBS and were fixed in 4% paraformaldehyde containing culture medium for 15min at 37°C. The nuclei were stained using DAPI. Cells were examined under fluorescence microscopy. For flow cytometry, the phagocytosis to BCG-p was performed as previously report [41]. Briefly, THP-1 cells were infected by GFP-expressing BCG-p for 4 h, then the cells were rinsed with PBS, next the cells were removed form plates and were measured with BD Accuri C6. The data was analyzed by the soft of Flowjo V10. Phagocytosis was determined by gating the cells and calculating the percent of cells that had taken up GFP-expressing BCG-p.

Colony-forming unit assay

Macrophages were infected in vitro with BCG-p at an MOI of 1, and incubated at 37˚C in a 5% CO2 humidified incubator. After infection 3 h, the cells were washed three times with PBS to remove extracellular bacteria and continued to culture with 50 μg/mL gentamycin for 1 h. Then the cells were rinsed three times with PBS, and were lysed by PBS containing 0.5% Triton X-100. The lysates were serially diluted and spotted on agar (7H10, 10% OADC) in triplicate. Colonies were counted after 2–3 weeks.

Statistical analysis

Experiments were repeated at least three times. All statistical analyses were performed using GraphPad Prism 8.0. The statistical comparison was evaluated with one- or two-way ANOVA. P < 0.05 was considered statistically significance.

Supporting information

S1 Fig. The raw image of protein expression of SLC39A7 in THP-1 cells.

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Click here for additional data file.^{(950.7KB, tif)}

S2 Fig. The raw image of protein expression of SLC39A7 in knockdown cells.

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Click here for additional data file.^{(1.1MB, tif)}

S3 Fig. The intracellular Zn concentration was measured using Fluozin-3^TM AM.

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S1 Table. The list of primers.

(DOCX)

Click here for additional data file.^{(13.6KB, docx)}

Acknowledgments

We thank colleagues Hui Ma and Kang Wu for helpful advice.

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

This work was supported a grant to K.-W. Wong by National Natural Science Foundation of China (Award Number: 81770010). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0235776.r001

Decision Letter 0

Rebecca A Hall

6 Mar 2020

PONE-D-20-03246

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

PLOS ONE

Dear Dr. Wong,

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Reviewer #1: The authors describe the link between the SLC39A7 zinc transporter knockdown and its effect on phagocytosis and M1 and M2 macrophage response. While this manuscript is covering an interesting area some of the conclusions are not in line with the results shown and the link between asthma and BCG is not discussed.

The manuscript has many English mistakes and is not fully formatted (1. Headline missing number, 5. Headline still includes ???). Some language is non-scientific (line 97 – partially rescue the situation). The statistics are only partially done and the p-values in the legend don’t match the ones show in the figures. The quality of the figures is rather poor with very low resolution which make the axis labels blurry. There are two supplementary figures which are not mentioned anywhere.

Explain why mRNA experiments were done with MOI of 10 when all other experiments were done with MOI of 5?

Figure1: No statistics on time point 24h, no indication of how many repeats were done, for p-values only ** and *** are mentioned in the legend which actually are not shown in the figure, the increase on SLC39A7 on protein level is not that obvious, a quantification of signal strength with image J would be beneficial.

Figure2: The decrease of proliferation is rather slow and there is no difference for the first 72h and only a small but significant increase after 96h. And yet adhesion studies omit this time point and only go until 72h. It is claimed that the addition of Zn and pyrithione rescues the failure in adhesion which can only be shown for 1 of the 2 SLC39A7 knockdown (KD-4) whereas KD-2 is unresponsive to the addition of zinc. There is mentioning of all THREE knockdown cell lines in figure legend 2 when only 2 were analysed. Statistic don’t match up again.

Figure3: No mentioning of sonicated vs non-sonicated anywhere in the in the text or legend (either take out the of the figures or explain in text).

Figure4: Shows the same before mentioned discrepancy between the two different knockdown cell lines. M2 marker Arg-1 was only increased in one of the two KDs. The authors picked the one that fits his theory best and did not discuss the unresponsiveness of KD-2. Same goes for IL-10 expression. Stats, p-values don’t match again. Stats, p-value

Figure5: TLR4 is already up before infection and is increased upon BCG phagocytosis. Address this phenomenon. Stats, p-value

Figure6: How many repeats?

Especially due to the different responses of the two KD cell lines this paper would benefit from measuring the actual zinc level of the knockdowns compared to NC by either ICP-MS/ICP-OES or by FluoZin (or similar) and flow cytometry. I also suggest measuring interleukin level directly by ELISA rather than extrapolate their increases by gene expression.

Reviewer #2: The authors of the article 'Zinc transporter SLC39A7 relieves zinc deficiency to suppress alternative 2 macrophage activation and impairment of phagocytosis' are providing interesting observations about the role the zinc transporter SLC39A7 on macrophage functions and polarization.

This manuscript is solely descriptive and doesn't provide insight about the molecular mechanisms under the control of SLC39A7. However, I think these observations are interesting and original. They bring a new set of evidences suggesting that the zinc has an important role in modulating the immune response and the host-pathogen interactions.

I think the manuscript could be improved by the following suggestions:

Major comments:

Line 63: SLC39A7 links with asthma. The reference is missing here. Is it https://www.jimmunol.org/content/197/2/655 ? Please provide the related reference.

Fig. 2: What is the difference between THP-1 cells and NC cells? Why are the THP-1 (which are a the original non-transfected cell line) not behaving as this non-target transfected NC cells but as the KD#2 and KD#4? This point is important as it suggests that KD#2 and KD#4 are proliferating at the same rate as conventional THP-1. However, the authors don’t stress this point and prefer to focus on the results obtained with the NC strain.

Line 89, Line 92, Fig. 2 B: considering the way this CCK8 assay is set-up, I think it is incorrect to write ‘cell viability’ here as the authors are in fact measuring the cell number (which can be affect the cell proliferation rate rather than the death rate) via the CCK8 assay.

Line 98 to 100: What is the timepoint of cell culture at which the SYTOX Green staining has been performed?

Legend Figure 2 C: What is the time point of culture at which the SYTOX Green staining has been performed?

Line 117, line 118, line 121, line 124, line 138: As the authors studied the transcript expression levels of genes associated with the pro-inflammatory (M1) and resolving (M2) macrophages, and not the actual protein expression from these genes, it would be more accurate to write that they analysed ‘the transcript expression levels of genes’ rather than ‘expression of markers’, ‘the surface protein CD11c’, ‘the expression of cytokines’, ‘expression of receptors Clec4e’.

Line 133: Once again I would advised to write ‘the transcript expression levels of Clec4e was reduced…’ as the authors are just using quantitative PCR for those experiments and not the actual protein levels.

Line 142-144, I don’t think the authors can come to the conclusion that ‘These results suggested that SLC39A7 played a role in the macrophage phagocytosis through mediating the expression of cell surface protein Clec4e in sensing microbial invasion’ as they don’t show any results related to differential protein expression of CLEC4e (Mincle) in their cell lines and didn’t try to rescue the phenotype observed by forcing CLEC4e expression in those cell lines or didn’t try to mimic the phenotype observed by blocking CLEC4e in their assay.

Results, Paragraph 5: Why are the author not describing in the results section the differences they observed for TLR4 gene expression (Figure 5). It is one of their major finding that could also explain the differences observed with the phagocytosis?

Line 179: ‘The expression of TLR4’, please add ‘gene’ as you are looking for the transcript and not the protein.

Line 196: ‘the pro-inflammatory cytokines TNF-a’, please add ‘the gene coding for ’ as you are looking for the transcript and not the protein. Same thing on line 197 for IL-10 and line 200 for IL-6.

Supplemental Figure: The authors are providing two supplemental figures, however there is no mention of these figures inside the result section of the manuscript.

Material and Methods, Phagocytosis Assay section: The authors report that they have performed some of the phagocytosis assay by flow cytometry. However, there is no obvious reference about these flow cytometry experiments in the rest of the manuscript.

Minor comments:

Introduction and Discussion: I think this manuscript would benefit from citing and discussing recent important works done on SLC39A7 in B cell development (doi: 10.1038/s41590-018-0295-8) and glycolysis (doi: 10.1371/journal.pone.0079316. eCollection 2013.) in the context of immune cells crosstalk and the importance of immuno-metabolism in macrophage functions and polarization.

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Reviewer #1: Yes: Claudia Simm

Reviewer #2: Yes: Guillaume E. Desanti

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PLoS One. 2020 Jul 9;15(7):e0235776. doi: 10.1371/journal.pone.0235776.r002

Author response to Decision Letter 0

21 Apr 2020

Point-to-point responses

1. Response to Reviewer #1

1) Q: manuscript is covering an interesting area some of the conclusions are not in line with the results shown and the link between asthma and BCG is not discussed. The manuscript has many English mistakes and is not fully formatted.

A: We thought carefully our results and conclusion, and agreed that our conclusion was improper, so we have corrected our conclusion according our experimental results (line 43-44 and line 150-151). We agreed that we made an inaccurate conclusion: ‘These results suggested that SLC39A7 played a role in the macrophage phagocytosis through mediating the expression of cell surface protein Clec4e in sensing microbial invasion.’ This statement is not replaced with the following statement:

Line 43-44: “Taken together, these results suggested that SLC39A7 played a role in the macrophage phagocytosis.”

In the introduction section, we also add some information about the link between asthma and BCG (line 73-75).

Line 73-75: “BCG is a vaccine to prevent tuberculosis and has also been used as a potent immunomodulator in the decades [17]. BCG has been reported that the protection against asthma [18].”

We went over our manuscript again three times and corrected many mistakes and inappropriate words. The statistics and figures were done again, and the information of supplementary figures (line 89, line 94, line 133-136, line 154-156 and the supporting information section) was added in the manuscript.

2) Q: Explain why mRNA experiments were done with MOI of 10 when all other experiments were done with MOI of 5?

A: We performed the mRNA experiments at 6 h after BCG-p infection; we increased the MOI to 10 for a higher host response to the BCG-p. In the other experiments, the BCG-p at MOI of 5 was used to display the difference between control cell and knockdown cell better.

3) Q: Figure1: No statistics on time point 24h, no indication of how many repeats were done.

A: We did mistakenly maked these errors in the statistics due to our negligence. We have reproduced Fig.1A and corrected the indistinctive description using Graphpad Prism 8.0 (line 420).

4) Q: Figure2: The decrease of proliferation is rather slow and there is no difference for the first 72h and only a small but significant increase after 96h. And yet adhesion studies omit this time point and only go until 72h.

A: The data of Fig.2B was analyzed again (line 426). At the 72 h in the proliferation assay, there is difference between control and SLC39A7 knockdown cell, but it was difficult to visualize the difference, we changed the graph type of Fig.2B and showed the difference between KDs and control (NC). Moreover, because all experiments were performed at 72 h after PMA stimulation, the THP-1 cells would become macrophages that incapable of proliferation, thus the adhesion assay was done only up to 72 h. We also corrected the word of ‘three’ in the figure legend (line 425).

5) Q: Figure3: No mentioning of sonicated vs non-sonicated anywhere in the in the text or legend (either take out the of the figures or explain in text).

A: We believed the result infection of non-sonicated BCG-p in Fig.3 was irrelevant and therefore was removed in the revised verison.

6) Q: Figure4: The authors picked the one that fits his theory best and did not discuss the unresponsiveness of KD-2. Same goes for IL-10 expression. Stats, p-values don’t match again. Stats, p-value.

A: We repeated our experiments at least three times, and got same trend. We presumed that the different adhesion of the two KD cells caused the discrepancy (line 98-100, Fig. 2D). KD-4 has a bigger defect in adhesion and a more responsiveness in cytokine productions. It is interesting to note that monocytes from systemic lupus erythematosus have defects in adhesion and produce elevated levels of IL-6, TNF-a, and IL-10 than healthy monocytes (Yi et al., 2010, Arch Immunol Ther Exp, PMID 20676786). Thus, the discrepancy between KD-2 and KD-4 could reflect a differential compensatory response by the KD cells. We also measured the intracellular concentration of Zn2+ by Fluozin-3TM AM using flow cytometry and found they were significantly decreased in the two KDs, but there was no difference between the two KDs (Figure S4). The statistics analysis was done again, and the p-value was corrected due to our careless (line 433-434).

7) Q: Figure5: TLR4 is already up before infection and is increased upon BCG phagocytosis. Address this phenomenon. Stats, p-value

A: The TLR4 signaling could be activated by extracellular inflammatory molecule HMGB1. The deficiency of SLC39A7 could induce cell death and lead to the release of HMGB1. Thus the increase of TLR4 mRNA could be induced by HMGB1 in knockdown SLC39A7 cells (line 183-186). The statistics analysis was done again, and the p-value was corrected due to our carelessness (line 449).

8) Q: Figure6: How many repeats?

A: All experiments were done at least three times. According to reviewer’s suggestion, we measured the intracellular zinc level by Fluozin-3 AM, and added the figure in supplemental section (Figure S4, line 154-156 ), the result showed that the intracellular Zn2+ concentration in KDs was significantly lower than that in control cells. We also performed ELISA assays for interleukin level at 48h after BCG-p infection (Figure S3, line 133-136), the results showed that proinflammatory cytokines TNF-α and IL-6 were significantly decreased in KDs at 48 hour after BCG-p infection. The mRNA of IL-6 was inconsistent with the protein, which could be caused by the improper expression of mRNA. ER is the place of mRNA translation; the knockdown of SLC39A7 could induce the UPR response, which leads the improper translation of mRNA.

2. Response to Reviewer #2

1) Q: Line 63: SLC39A7 links with asthma. The reference is missing here. Is it https://www.jimmunol.org/content/197/2/655 ? Please provide the related reference.

A: Thanks for your kindly reminder. We added the reference in the Introduction (line 71, ref.16).

2) Q: Fig. 2: What is the difference between THP-1 cells and NC cells? However, the authors don’t stress this point and prefer to focus on the results obtained with the NC strain.

A: NC cells are THP-1 cells transfected with control plasmid containing no target sequence. Because the KD#2 and KD#4 cells are THP-1 cells transfected with plasmid containing target sequence, we thought that we should use NC cells as control cells. The phagocytosis efficiency of the THP-1 cells and NC cells are comparable, so in the other experiments we performed assays only in NC cells. We checked the proliferation rate of all cells three times and found that the proliferation rate of NC was faster than others, but we don’t know how it caused, it may be caused by the different cleaved position by Cas9.

3) Q: it is incorrect to write ‘cell viability’ here as the authors are in fact measuring the cell number via the CCK8 assay.

A: We corrected the word of “cell viability” with cell proliferation rate (line 95, line 98).

4) Q: Line 98 to 100: What is the timepoint of cell culture at which the SYTOX Green staining has been performed?

A: We checked the cell death with SYTOX Green staining at 72 h after PMA stimulation (line 103).

5) Q: Legend Figure 2 C: What is the time point of culture at which the SYTOX Green staining has been performed?

A: We performed the SYTOX Green staining at 72 h after PMA stimulation, and added the timepoint in the figure 2C (line 427-430).

6) Q: As the authors studied the transcript expression levels of genes associated with the pro-inflammatory (M1) and resolving (M2) macrophages, it would be more accurate to write that they analysed ‘the transcript expression levels of genes’ rather than ‘expression of markers’.

A: We corrected the words ‘the expression of markers’ with ‘the mRNA levels of genes in all corresponsive positions (line 123, line 125, and line 126).

7) Q: Line 133: Once again I would advised to write ‘the transcript expression levels of Clec4e was reduced…’

A: We changed the word ‘expression’ with ‘the mRNA level’ (line 146).

8) Q: Line 142-144, I don’t think the authors can come to the conclusion that ‘These results suggested that SLC39A7 played a role in the macrophage phagocytosis through mediating the expression of cell surface protein Clec4e in sensing microbial invasion’.

A: We have corrected our conclusion and deleted the words ‘mediation the expression of Clec4e in sensing microbial invasion’ (line 43-44, line 150-151).

Line 43-44: “Taken together, these results suggested that SLC39A7 played a role in the macrophage phagocytosis.”

Line 150-151: “These results suggested that SLC39A7 played a role in the macrophage phagocytosis; the deficiency of SLC39A7 could reduce the transcription level of Clec4e.”

9) Q: Results, Paragraph 5: Why are the author not describing in the results section the differences they observed for TLR4 gene expression (Figure 5). It is one of their major finding that could also explain the differences observed with the phagocytosis?

A: Thank you for your kindly suggestion, we neglected the results of TLR4 gene expression due to our focus on the Clec4e gene expression. In the new manuscript, we added the analysis of TLR4 gene expression (line 146 and line 228-231).

Line 146: “The results showed that the mRNA level of receptors Clec4e (Mincle) was significantly decreased and TLR4 mRNA was increased in SLC39A7 knockdown cells (Fig.5).”

Line 183-186: “The expression of TLR4 was slightly increased in SLC39A7 knockdown cells. The phenomenon could be caused by the cell death in SLC39A7 knockdown cells. The deficiency of SLC39A7 leads to the cell death and releases the extracellular inflammatory factor HMGB1, which could activate TLR4 expression [29].”

10) Q: Line 179: ‘The expression of TLR4’, please add ‘gene’ as you are looking for the transcript and not the protein.

A: Thank you for your advice again, we corrected ‘the expression of TLR4’ with ‘the TLR4 mRNA level’ (line 146).

11) Q: Line 196: ‘the pro-inflammatory cytokines TNF-a’, please add ‘the gene coding for ‘. Same thing on line 197 for IL-10 and line 200 for IL-6.

A: We corrected all ‘the expression of’ with ‘the gene expression of’ in the RT-PCR results (line 129, line 130, and line 131).

12) Q: there is no mention of supplement figures inside the result section of the manuscript.

A: We added the supplemental figures at their corresponsive positions in the manuscript (line 89, line 94, line 136, line 156, and line 291-296).

13) Q: there is no obvious reference about these flow cytometry experiments in the rest of the manuscript.

A: In the Figure 3B and 6A, we performed the phagocytosis assay by flow cytometry. We also added a reference (ref.42, Kordon AO, et al., 2017, Front Microbiol. doi: 10.3389/fmicb.2017.02638.) in the method of phagocytosis assay.

14) Q: Minor comments: I think this manuscript would benefit from citing and discussing recent important works done on SLC39A7 in B cell development (doi: 10.1038/s41590-018-0295-8) and glycolysis (doi: 10.1371/journal.pone.0079316. eCollection 2013.)

A: Thank you for the suggestion. We added discussion about the SLC39A7 had reported in B cell development and glycolysis, all these results hinted the importance of SLC39A7 in immune-metabolism (line 65-68 and line 214-218).

Line 65-68 in Introduction: “Recent data shows that SLC39A7 is implicated in glucose metabolism and glycemic control in skeletal muscle cells [13, 14]. In the immune cells, it was reported that SLC39A7 has essential role in B cell development; the intact of SLC39A7 was required for BCR signaling [15].”

Line 214-218 in Discussion: “Recent data shows that SLC39A7 is implicated in glucose metabolism and glycemic control in skeletal muscle cells [13, 14]. SLC39A7 has essential role in B cell development, SLC39A7 was required for BCR signaling [15]. All these observations showed that SLC39A7 played an essential role in the host immune metabolism. The function of SLC39A7 should be further investigated.”

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PLoS One. doi: 10.1371/journal.pone.0235776.r003

Decision Letter 1

Rebecca A Hall

29 Apr 2020

PONE-D-20-03246R1

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

PLOS ONE

Dear Wong,

We would appreciate receiving your revised manuscript by Jun 13 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
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We look forward to receiving your revised manuscript.

Kind regards,

Rebecca A Hall

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

Thank you for submitting a revised version of your manuscript. I can see that you have addressed most of the comments made by the Reviewers but there are still a few points of clarification required before the manuscript can be submitted.

The main finding of the study appears to be that knockdown of SLC39A7 reduces phagocytosis of BCG, through the depletion of intracellular Zn levels, which potentially results in reduced expression of Clec4e. However, this is not clear from the text. Some sections of the text imply that SLC39A7 is directly involved in the phagocytosis of BGC (i.e. line 167-168), which I do not think the authors mean to imply. I think this is a communication issue, rather than an issue with the interpretation of the data, and I strongly suggest that the author’s seek help from a native speaker. Even with the additional proof reading between the original submission and the revised version many English mistakes remain that affect the understanding of the manuscript.

Fig2D: Zn does not restore adhesion in KD2, only KD4. This was raised by Reviewer 1, and addressed in the rebuttal letter, but no mention was made in the actual manuscript. An explanation in the manuscript is required. Did you measure intracellular Zn after the addition of extracellular Zn to check that intracellular Zn levels were restored in both cases?

Were the other phenotypes (cytokine expression and PRR expression) restored by the supplementation of exogenous Zn?

Fig5: why is there no data for the Clec4e control?

Line 167-168: We found that SLC39A7 mediated the phagocytosis of BCG by THP-1 cells. Do you really mean this?

Line 209: Unregulated, should this be up-regulated?

Figure 6 could be combined with Fig 3

[Note: HTML markup is below. Please do not edit.]

PLoS One. 2020 Jul 9;15(7):e0235776. doi: 10.1371/journal.pone.0235776.r004

Author response to Decision Letter 1

18 Jun 2020

1) Q：The main finding of the study appears to be that knockdown of SLC39A7 reduces phagocytosis of BCG, through the depletion of intracellular Zn levels, which potentially results in reduced expression of Clec4e. However, this is not clear from the text. Some sections of the text imply that SLC39A7 is directly involved in the phagocytosis of BCG (i.e. line 167-168), which I do not think the authors mean to imply.

A：Thank for your kindly advice. The sentence “SLC39A7 mediated the phagocytosis to BCG-p” was now replaced with “SLC39A7 played a role in the phagocytosis of BCG by THP-1 cells (line 174)”.

We have edited the manuscript extensively and corrected many instances of poor usage of English. They are all highlighted in RED colors.

2) Q：Fig2D: Zn does not restore adhesion in KD2, only KD4. This was raised by Reviewer 1, and addressed in the rebuttal letter, but no mention was made in the actual manuscript. An explanation in the manuscript is required. Did you measure intracellular Zn after the addition of extracellular Zn to check that intracellular Zn levels were restored in both cases?

A: We performed new experiments and found that zinc supplementation substantially increased intracellular Zn2+ levels in KD2 and KD4 (Fig. S3). This elevated intracellular zinc level by supplementation was not sufficient to restore the adhesion defect in KD2. Thus, the adhesion defect in KD2 could be independent of a SLC39A7 defect that could be restored by zinc supplementation.

The following statements were added to explain the unresponsive phenotypes in KD2 (line 207-214 in Discussion):

“One of the KD cells KD-2 did not exhibit defects in several cytokine expressions (Fig. 4A-B), and was unresponsive zinc supplementation. We presumed that the different adhesion of the two knockdown cells caused the discrepancy. KD-4 has a bigger defect in adhesion and a more responsiveness in cytokine productions. It is interesting to note that monocytes from systemic lupus erythematosus have defects in adhesion and produce elevated levels of IL-6, TNF-a, and IL-10 than healthy monocytes [38]. Thus, the discrepancy between KD-2 and KD-4 could reflect a differential compensatory response by the KD cells.”

Note: In the new sets of experiments only upregulation of the expression of M2 macrophage marker CD206 in response to SLC39A7 knockdown was reproduced and this upregulation could be reversed by zinc supplementation. mRNA expression of M2 macrophage marker genes Arg-1 and Ym-1 was either unchanged in knockdown cells or unresponsive to zinc supplementation. It is notable that the CD206 upregulation was not seen in KD2, consistent with its unresponsiveness. Collectively, the SLC39A7-dependent regulation of M2 macrophage marker CD206 expression (rescued by zinc supplementation) indicated that loss of SLC39A7 skewed macrophages towards alternative M2 activation.

In this second version of revised manuscript, we have one phenotype that could not be reproduced. Expression of IL-6 was down-regulated in both BCG-p infected KD cells in two of our recent new experiments, in contrast to the previous revision where expression of IL-6 was up-regulated (Fig. 5A). But since this IL-6 expression down-regulation could not be restored by zinc supplementation (Fig. 5A), this down-regulation phenotype could be unrelated to the zinc transporter function of SLC39A7. Despite this observation, the release of IL-6 proteins was dependent on the zinc transporter function of SLC39A7 (Fig. 5B), indicating that SLC39A7 likely regulates IL-6 post-translationally. Importantly, this result is consistent with the importance of SLC39A7 in supporting a proinflammatory profile for classically activated macrophage.

3) Q：Were the other phenotypes (cytokine expression and PRR expression) restored by the supplementation of exogenous Zn?

A: We also assayed the PRR (Clec4e and TLR4) mRNA expression and found that the mRNA levels of Clec4e and TLR4 restored by the supplementation of Zn2+ (line 147-149, Fig.6). We assayed the mRNA expression of cytokines (line 135-138, Fig.4, Fig.5), and found that the mRNA levels of cytokines could not restored by the supplementation of Zn2+, however the level of TNF-α and IL-6 proteins in the supernatants were restored by supplementation of exogenous Zn2+ (line 135-139, Fig.5B). IL-10 was undetectable using ELISA in uninfected and infected THP-1 cells.

4) Q：Fig5: why is there no data for the Clec4e control?

A: The expression of Clec4e in uninfected cells was negligible in comparison with that in BCG-p-infected cells where the expression of Clec4e increased dramatically, making it looks like there is no data for the Clec4e control. We reformatted the figure and included the data with the conditions of supplementation of Zn2+ (Fig.6).

5) Q：Line 167-168: We found that SLC39A7 mediated the phagocytosis of BCG by THP-1 cells. Do you really mean this?

A: We replaced the text with the following sentence “SLC39A7 played a role in the phagocytosis of BCG by THP-1 cells” (line 162).

6) Q：Line 209: Unregulated, should this be up-regulated?

A: Indeed, “up-regulated” was now used (line 207).

7) Q：Figure 6 could be combined with Fig 3

A: Fig 6 is now combined with Fig 3.

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PLoS One. doi: 10.1371/journal.pone.0235776.r005

Decision Letter 2

Rebecca A Hall

23 Jun 2020

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

PONE-D-20-03246R2

Dear Dr. Wong,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Rebecca A Hall

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Please check the final version for grammatical errors.

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0235776.r006

Acceptance letter

Rebecca A Hall

25 Jun 2020

PONE-D-20-03246R2

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

Dear Dr. Wong:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Rebecca A Hall

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. The raw image of protein expression of SLC39A7 in THP-1 cells.

(TIF)

Click here for additional data file.^{(950.7KB, tif)}

S2 Fig. The raw image of protein expression of SLC39A7 in knockdown cells.

(TIF)

Click here for additional data file.^{(1.1MB, tif)}

S3 Fig. The intracellular Zn concentration was measured using Fluozin-3^TM AM.

(TIF)

Click here for additional data file.^{(57.6KB, tif)}

S1 Table. The list of primers.

(DOCX)

Click here for additional data file.^{(13.6KB, docx)}

Attachment

Submitted filename: Rebuttal letter 20200419_KWedited2.docx

Click here for additional data file.^{(35.3KB, docx)}

Attachment

Submitted filename: rebuttal letter 20200618_SECOND REVISION.docx

Click here for additional data file.^{(19.5KB, docx)}

Data Availability Statement

All relevant data are within the manuscript and its Supporting Information files.

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PERMALINK

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

Wenyan Xie

Qinghua Xue

Liangfei Niu

Ka-Wing Wong

Roles

Abstract

Introduction

Results

1. Expression of SLC39A7 increased in BCG infected macrophages

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

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

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

3. Knockdown of SLC39A7 disrupted phagocytosis of BCG-p by THP-1 cells

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

4. The mRNA expression of markers and cytokines associated with M1/M2 macrophage activation in SLC39A7-knockdown THP-1 cells

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

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

5. The expression of Clec4e was reduced in SLC39A7-knockdown cells

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

Discussion

Materials and methods

Cell culture

Generation of Knockdown cell line with CRISPR/Cas9

Cell Counting Kit-8 (CCK8) assay

Cell adherence assay

Bacterial strains and cultures

Reverse transcription and quantitative real-time PCR

ELISA

Phagocytosis assay

Colony-forming unit assay

Statistical analysis

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Rebecca A Hall

Roles

Author response to Decision Letter 0

Decision Letter 1

Rebecca A Hall

Roles

Author response to Decision Letter 1

Decision Letter 2

Rebecca A Hall

Roles

Acceptance letter

Rebecca A Hall

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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