NR1D1 ameliorates Mycobacterium tuberculosis clearance through regulation of autophagy

Abstract

NR1D1 (nuclear receptor subfamily 1, group D, member 1), an adopted orphan nuclear receptor, is widely known to orchestrate the expression of genes involved in various biological processes such as adipogenesis, skeletal muscle differentiation, and lipid and glucose metabolism. Emerging evidence suggests that various members of the nuclear receptor superfamily perform a decisive role in the modulation of autophagy. Recently, NR1D1 has been implicated in augmenting the antimycobacterial properties of macrophages and providing protection against Mycobacterium tuberculosis infection by downregulating the expression of the IL10 gene in human macrophages. This antiinfective property of NR1D1 suggests the need for an improved understanding of its role in other host-associated antimycobacterial pathways. The results presented here demonstrate that in human macrophages either ectopic expression of NR1D1 or treatment with its agonist, GSK4112, enhanced the number of acidic vacuoles as well as the level of MAP1LC3-II, a signature molecule for determination of autophagy progression, in a concentration- and time-dependent manner. Conversely, a decrease in NR1D1 in knockdown cells resulted in the reduced expression of lysosomal-associated membrane protein 1, LAMP1, commensurate with a decrease in the level of transcription factor EB, TFEB. This is indicative of that NR1D1 may have a regulatory role in lysosome biogenesis. NR1D1 being a repressor, its positive regulation on LAMP1 and TFEB is suggestive of an indirect byzantine mechanism of action. Its role in the modulation of autophagy and lysosome biogenesis together with its ability to repress IL10 gene expression supports the theory that NR1D1 has a pivotal antimycobacterial function in human macrophages.

Abbreviations

3-MA

3-methyladenine

AO

acridine orange

ARNTL/BMAL1

aryl hydrocarbon receptor nuclear translocator-like

AVOs

acidic vesicular organelles

CFU

colony forming unit

LAMP1

lysosomal-associated membrane protein 1

MAP1LC3/LC3

microtubule-associated protein 1 light chain 3

MDC

monodansylcadaverine

PFA

paraformaldehyde

PIK3C3/VPS34

phosphatidylinositol 3-kinase, catalytic subunit type 3

PMA

phorbol 12-myristate 13-acetate

SD

standard deviation

TFEB

transcription factor EB

ULK1

unc-51 like autophagy activating kinase 1

V-ATPase

vacuolar-type H⁺ ATPase

VPS11

vacuolar protein sorting 11 homolog (S. cerevisiae).

Introduction

The nuclear receptor super family includes various ligand-activated and orphan nuclear receptors that have varied functions.^1,2 NR1D1/Rev-Erba belongs to the adopted orphan nuclear receptor family with heme as its endogenous ligand.³ NR1D1 generally behaves as a transcriptional repressor, sometimes independently but often through recruitment of corepressor proteins, such as NCOR (nuclear receptor corepressor) and HDAC3 (histone deacetylase 3). NR1D1 is expressed in and modulates the function of various cell types including adipose, vascular smooth muscle, skeletal muscle, liver, heart, brain, and immune cells such as T-cells and macrophages.⁴ In the past decade NR1D1 has been reported to be a critical modulator in the regulation of the differentiation of cells such as adipocytes and smooth muscle. It has also been shown to be involved in maintaining metabolic homeostasis through hepatic lipid and glucose metabolism.^5-9 Our recent report establishes its role in ameliorating Mycobacterium tuberculosis clearance by direct repression of the human IL10 gene.¹⁰ Recent reports, including our study on the role of NR1D1 in infection and inflammation, suggest that NR1D1 behaves as an equilibrist whereby it finetunes the regulatory pathways in order to maintain the equilibrium in cellular processes.^10-12

Many proteins are involved in the expulsion of bacterium from the host. These include CLEC7A/dectin-1, TLRs (toll-like receptors), dendritic cell-specific CD209/DC-SIGN, soluble C-type lectins such as SFTPA (surfactant protein A), SFTPD and MBL (mannose-binding lectin), VDR (vitamin D [1,25-dihydroxyvitamin D3] receptor), and NR1H/LXR.^13-18 Macrophage antimicrobial properties are guided by phagosomal maturation, acidification of phagosomes, and activation of the NADH oxidase CYBB/NOX2 and NOS2/iNOS, as well as antimicrobial peptides and degradative proteins.^19-22 After phagocytosis, primary stimulation of antimicrobial activity is provided by the secretion of proinflammatory cytokines TNF, IL1B, IL6, and IL12.^23-25 The T-cell-driven immunity amplifies the ability of macrophages to kill and digest bacteria by inducing Th1 response and augmenting its cytotoxic activity.^26,27

In order to survive within the host system and counter frontline drug regimens, Mycobacterium tuberculosis has profoundly evolved resulting in the generation of multidrug-resistant (MDR) and extremely drug-resistant (XDR) strains.^28,29 New insights into macrophage-pathogen interactions and their roles in Mycobacterium tuberculosis pathogenesis can suggest exciting new therapeutic targets for host-specific adjunct therapies.^30,31 Autophagy, in particular macroautophagy, has been widely explored as an evolutionarily conserved catabolic process for cell viability and precise functioning of physiological processes.^32,33 Moreover, it has now been accepted as a crucial host-defense mechanism against Mycobacterium tuberculosis by spanning innate and adaptive immune functions.^34,35 Autophagy has been shown to be regulated at the transcriptional level, suggesting the possibility of finding genomic regulators that are amenable to pharmacological modulation.^36-38 On the basis of recent reports on the roles of NR1D1 in infection and inflammation, we sought to determine if NR1D1-mediated amelioration of Mycobacterium tuberculosis clearance is a result of its modulation of autophagy. In this study we have demonstrated that NR1D1 not only induces autophagy but also modulates expression of TFEB expression in human macrophages. GSK4112, a synthetic agonist of NR1D1, also induces autophagy in human macrophages. In addition, we observed a change of expression in some critical genes involved in lysosome biogenesis, which adds through an indirect pathway to the effect of NR1D1 on elevating autophagy. This enhanced combinatorial effect of autophagy and lysosome biogenesis further supports the role of NR1D1 in the clearance of Mycobacterium tuberculosis.

Results

Modulation of MAP1LC3 maturation in response to NR1D1 overexpression and activation by GSK4112 in macrophages

NR1D1 has been implicated in the regulation of various metabolic, infectious, and circadian processes. Only recently has it been shown to also have a role in autophagy in skeletal muscle cells.³⁹ MAP1LC3-I/LC3-I is cytosolic in nature. However, upon autophagic induction, it is conjugated to phosphatidylethanolamine giving rise to a lipidated form, LC3-II, which then localizes to the membranes of autophagic structures, phagophores, the precursors to autophagosomes. To explore the role of NR1D1 in immune cells, we studied a prototype cell line of human macrophage THP-1 cells treated with phorbol 12-myristate 13-acetate (PMA). We ectopically overexpressed Flag-tagged human NR1D1 in macrophages and investigated its ability to modulate LC3-II levels in absence or presence of bafilomycin A₁ (400 nM). Unlike what is seen in skeletal muscle, we observed an increase in LC3-II levels in these THP-1 macrophages, which suggests positive regulation of autophagy by NR1D1 (Fig. 1A). Overexpression of NR1D1 was confirmed through western blotting in THP-1 macrophages (Fig. S1A). Autophagy-regulated protein SQSTM1 serves a good marker to assess the phenomenon. We also monitored the expression of SQSTM1 in the same set and observed that there was a degradation of SQSTM1 in p-Flag-NR1D1 transfected cells while there was an accumulation of SQSTM1 protein in p-Flag-NR1D1 transfected cells treated with bafilomycin A₁. This indicates that there is an induction of autophagy upon overexpression of Flag-tagged-NR1D1.

Figure 1.

NR1D1 overexpression and activation by its agonist GSK4112 induces autophagy in macrophages. Western blot and densitometric analysis of (A) LC3-II and SQSTM1 protein levels in THP-1 macrophage cells overexpressing Flag-tagged NR1D1 in presence or absence of bafilomycin A₁ (400 nM), (B) Dose-dependent increase in LC3-II accumulation in macrophage cells treated with GSK4112 for 12 h, (C) Kinetics of LC3-II accumulation in macrophage cells treated with 20 µM GSK4112. The fold change in LC3-II levels compared to that of the control is shown under the blots. See also Figures S1 and S2. Changes in LC3-I and LC3-II protein levels are illustrated by LC3-II/ACTB (A, B and C) ratios in bar graphs as in insets. Data are mean ± SD from 4 independent experiments. *, P < 0.05 as compared to control or as indicated.

Given the observed increase in autophagic protein upon overexpression of NR1D1, we next examined the dose-dependent effects of GSK4112, an agonist of NR1D1, on LC3-II accumulation (Fig. 1B). It is well established that GSK4112 augments the repressive activity of NR1D1 by enhancing corepressor recruitment. THP-1 macrophages treated with 0 to 30 µM of GSK4112 for 12 h displayed a dose-dependent increase in LC3-II (Fig. 1B). A time kinetics experiment was then performed for 24 h with the agonist (GSK4112, 20 µM). A rapid increase in LC3-II maturation was seen between 4 and 24 h of incubation (Fig. 1C). GSK4112 activity was assessed by real-time PCR analysis of NR1D1 target genes IL10 and ARNTL/BMAL1 in THP-1 macrophages (Fig. S1B). We also investigated this phenomenon in primary human macrophage and murine macrophage cell line, RAW264.7, and observed a similar positive regulation of autophagy by NR1D1 (Fig. S2A). Two other human origin cell lines, HEK293 and HepG2, were evaluated for autophagic induction by GSK4112, and a slight modification in LC3-II protein level was observed (Fig. S2B). Treatment of GSK4112 to THP-1 macrophages in ATG5 and BECN1/Beclin 1 knockdown backgrounds did not affect the induction of autophagy, however, an increase in expression of LC3-I was observed indicating that NR1D1 may affect MAP1LC3 expression (Fig. S2C). The knockdown efficiency of ATG5 siRNA and BECN1 siRNA were assessed by real-time PCR analysis (Fig. S2C).

NR1D1 overexpression and activation by GSK4112 induces autophagy in human macrophages

Since LC3-II accumulation can be a result of the inhibition of the degradation of autophagosomes rather than an increase in autophagic flux, we treated macrophage cells with GSK4112 alone or in combination with 3-methyladenine (3-MA) or bafilomycin A₁ (Fig. 2A). An inhibitor of autophagy, 3-MA blocks type III phosphatidylinositol 3-kinase (PIK3C3/VPS34), which is essential for the initiation of autophagy through recruitment of other ATG proteins to the phagophore.^40,41 Bafilomycin A₁ prevents maturation of autophagic vacuoles by inhibiting vacuolar-type H⁺ ATPase (V-ATPase), thereby preventing degradation of autophagosomes.^42-44 Notably, 3-MA (10 mM) treatment in combination with GSK4112 (20 µM) abrogated, whereas bafilomycin A₁ (400 nM) in combination with GSK4112 (20 µM) resulted in a significant increase, in the levels of LC3-II in comparison to treatment with GSK4112 alone (Fig. 2A). This suggests that the increase in LC3-II induction by GSK4112 is due to an increase in autophagic flux.

Figure 2.

GSK4112 induced autophagic flux in human macrophages. (A) Western blot and densitometric analysis of LC3-II accumulation upon treatment with 20 µM GSK4112 and pretreatment with either 10 mM 3-MA or 400 nM bafilomycin A₁. (B) Flag-tagged NR1D1 expressing THP-1 macrophage and (C) GSK4112-treated macrophages were immunostained for LC3 in the presence or absence of 3-MA. (D) PMA-differentiated THP-1 cells stably expressing GFP-LC3 were treated with 20 µM GSK4112 (agonist), 20 µM SR8278 (antagonist), 10 mM 3-MA, 400 nM bafilomycin A₁, 100 nM wortmannin alone or in combination; and GFP-LC3 puncta formation was observed. Images are representative of 5 independent replicates. The number of GFP-LC3-II puncta in each cell was counted for 100 cells in each group. Quantification of the percentage of cells with LC3 puncta and LC3 puncta per cell is represented as mean ± SD from 3 independent experiments each performed in triplicates or as indicated. *, P< 0.05 as compared to control or as indicated.

Autophagosome formation can also be observed by confocal microscopy. Therefore, we performed immunostaining of LC3 protein after ectopic overexpression of Flag-tagged NR1D1 (Fig. 2B) or in the presence of GSK4112 treatment (Fig. 2C), alone or in combination with 3-MA. In the agonist-treated cells and the cells overexpressing NR1D1, there was an enhanced appearance of green fluorescent dots of immunostained LC3. The change in fluorescent intensity was quantified and showed a significant increase over the control in both samples. A similar pattern was not observed with 3-MA treatment, indicating an increase in autophagy flux. Confocal microscopy of THP-1 cells stably expressing GFP-LC3 in the presence of agonist or antagonist also confirmed of the positive regulation of autophagy by NR1D1 (Fig. 2D). Autophagy inhibitors wortmannin and bafilomycin A₁ block autophagy by inhibiting autophagosome formation and autolysosome degradation respectively. Treatment of GSK4112 with wortmannin (100 nM) resulted in less puncta whereas the number of punta increased significantly in cells treated with GSK4112 in presence of bafilomycin A₁ (400 nM). Treatment of starvation-induced cells with SR8278 (20 µM), antagonist of NR1D1, showed less number of puncta. Taken together the data confirmed that NR1D1 plays a crucial role in autophagy regulation.

NR1D1 activation by GSK4112 stimulates autophagy induction and modulation in lysosomal biogenesis in human macrophages

Development of acidic vesicular organelles (AVOs) in cell cytoplasm is one of the hallmarks of autophagy. To ascertain the role of NR1D1 in this process, acridine orange (AO) staining was performed in cells after GSK4112 treatment (Fig. 3A). This showed an increase in the number of AVOs in cells after agonist treatment compared to that in control cells. The addition of 3-MA significantly, but not completely, abrogated the increase in GSK4112-induced AVOs thus confirming the importance of NR1D1 in autophagy induction and suggesting that the phenomenon is governed by other pathways as well.

Figure 3.

GSK4112 stimulates autophagosome and lysosome formation in human macrophage cells. THP-1 macrophage cells were treated with 20 µM GSK4112 in the presence or absence of 10 mM 3-MA for 12 h followed by (A) AO and (B) MDC staining for acidic compartments. (C) Confocal microscopy was performed on THP-1 macrophages treated with either 20 µM GSK4112 alone or in combination with 3-MA or SR8278 for 12 h (n = 3) and stained with LysoTracker Red. (D) FACS analysis of LAMP1 (late endosomal/lysosomal marker) was performed on THP-1 macrophages treated with either 20 µM GSK4112 in presence or absence of 10 mM 3-MA or 20 µM SR8278 for 12 h. See also Figures S3 and S4. Images are representative of 5 independent replicates. The numbers of AVOs and LysoTracker Red puncta in each cell were counted in 100 cells in each group. Scale bar: 10 μm. Number of percent positive cells for LAMP1 was determined by flow cytometry comparing the percentage of cells stained, in the treatment conditions and unstained cells in the control. Data are mean ± SD from independent 3 to 5 experiments each performed in triplicates as indicated. *, P < 0.05 as compared to control or as indicated.

Additionally, when we performed monodansylcadaverine (MDC) staining, which is specific for acidic compartments, a similar result was observed (Fig. 3B). Data from these experiments are in agreement and suggest that NR1D1 induces increase in acidic compartments. In order to verify this, we performed LysoTracker Red staining and analyzed cells by confocal microscopy (Fig. 3C) and flow cytometry (Fig. S3) in the presence of agonist (GSK4112) and antagonist (SR8278). LysoTracker Red dye also stains acidic vesicles. There were more red puncta and LysoTracker Red staining in cells treated with GSK4112, illustrating an increase in acidic compartments (Fig. 3C). Further, upon treatment of 3-MA and GSK4112, a slight decrease in puncta formation was observed indicating that GSK4112 has its activating effect in lysosomal formation also. Antagonist treatment led to a reduction in red puncta and reduced LysoTracker Red staining, illustrating a decrease in acidic vesicles (Fig. 3C). We quantified the retention of LysoTracker Red by flow cytometry in cells treated with agonist and found 2-fold more lysosomes than the control cells which reduced significantly in cells treated with antagonist (Fig. S3). The above experiments are only indicative and therefore we also performed intracellular staining of a late endosomal/lysosomal marker, LAMP1 (lysosomal-associated membrane protein 1) in cells treated with GSK4112, which demonstrated an accumulation of LAMP1-bearing lysosomes in the cytosol of the cells by both FACS (Fig. 3D) and confocal imaging (Fig. S4).

NR1D1 modulates the expression of TFEB

TFEB (transcription factor EB), a bHLH-leucine zipper transcription factor, is known as a master transcription regulator, which coordinates lysosome formation and function in addition to regulating autophagic transcription during starvation.^45,46 Since there was an elevation in the number of LysoTracker Red-stained lysosomes in cells with increased NR1D1 activity, we were intrigued to assess the role of NR1D1 in TFEB modulation. We studied expression of TFEB in NR1D1 knockdown macrophage cells and cells overexpressing Flag-NR1D1 in NR1D1 knockdown background cells by western blot analysis and found that the level of TFEB protein was reduced in NR1D1 knockdown cells in comparison to that in the scramble control whereas an increase in expression of TFEB was observed in cells rescued with NR1D1 overexpression (Fig. 4A). We also compared the levels of LC3-II in order to corroborate the effect in terms of autophagy. In the presence of NR1D1 knockdown, there was a minimal change in the level of LC3-II, however an increase in LC3-II level observed upon ectopic expression of Flag-NR1D1 suggests that both the autophagy and lysosome biogenesis processes are impaired (Fig. 4A). Starvation induces TFEB activation, and upregulation of its downstream target genes (MAPLC3B, VPS11, VPS18, ATG9B, LAMP1, etc.) is through its localization to the nucleus.⁴⁵ LAMP1 is a known late-endosomal marker that is regulated by TFEB. To investigate the downstream effect of TFEB modulation on lysosome biogenesis in NR1D1 knockdown cells in addition to cells rescued with overexpression of Flag-NR1D1, we assessed LAMP1 protein expression by western blot analysis and found it to be reduced in NR1D1 knockdown cells (Fig. 4B). Also, the protein level of LAMP1 was enhanced in cells with ectopic expression of Flag-NR1D1. siRNA knockdown efficiency for NR1D1 was confirmed by western blot analysis and real-time PCR (Fig. 4C). Therefore, we next assessed LC3-II levels in normal and starved knockdown NR1D1 cells with and without bafilomycin A₁. The downregulation of TFEB level in the knockdown NR1D1 cells correlated with a decrease in LC3-II levels in starved cells both with and without bafilomycin A₁ treatment. This clearly indicates that NR1D1 comodulates biogenesis of autophagosomes and lysosomes (Fig. 4D).

Figure 4.

NR1D1 knockdown in THP-1 macrophage cells modulates expression of genes responsible for autophagy, lysosome biogenesis or lysosomal acidification. Western blot and densitometric analysis of (A) TFEB and LC3-II (B) LAMP1 protein levels in cells with NR1D1 knockdown (NR1D1 siRNA) and cells rescued by NR1D1 overexpression in NR1D1 knockdown background. (C) Real-time PCR and western blot analysis of the knockdown efficiency of NR1D1 in cells. (D) LC3-II and TFEB protein analysis in NR1D1 knockdown cells cultured in normal media, starvation media, and starvation media containing bafilomycin A₁ (400 nM for 4 h). (E) Quantitative real time PCR analysis of the published set of genes essential for autophagy and lysosome biogenesis and acidification. Data are mean ± SD from independent 3 to 5 experiments each performed in triplicates as indicated. *, P < 0.05 as compared to control or as indicated.

To further validate the effect of NR1D1 knockdown in human macrophage on the genes regulating autophagy and lysosome biogenesis, we performed quantitative real-time PCR of selected set of genes (18 genes) that have been widely described in literature. Among the selected genes known to regulate autophagy and lysosome biogenesis, 11 genes (LAMP1, ULK1, VPS18, ATG9, TFEB, LC3A, LC3B, GABARAPL1, PIK3C3, ATG7 and VPS11) exhibited a significant decrease in expression in the NR1D1 knockdown background in comparison to control (Fig. 4E).

NR1D1 activation by GSK4112 augments clearance of Mycobacterium tuberculosis from human macrophages by increasing autophagic flux and TFEB-associated pathways

Our previous study has demonstrated the clearance of Mycobacterium tuberculosis upon overexpression of NR1D1 in human macrophages through direct gene repression of the human IL10 gene.¹⁰ However, this clearance may not be the outcome of a solitary mechanism but rather from a group of pathways converging to the same endpoint. The observation in the present study of an increase in both autophagic flux and modulation in lysosome biogenesis led us to examine the relationship between these processes. Our aim was to enumerate the number of autophagosomes and lysosomes. This was possible with the use of bafilomycin A₁, which blocks fusion of autophagosomes with lysosomes and thus allows independent enumeration. We performed immunostaining of LC3 protein and LAMP1 in Mycobacterium tuberculosis H37Rv-infected cells treated with GSK4112 alone and in combination with bafilomycin A₁ and determined the number of autophagosomes (Fig. 5A) and lysosomes and autolysosomes (Fig. 5B), both of which were increased significantly in comparison to infected cells without treatment of agonist. GSK4112 caused a significant increase in the colocalization of Mycobacterium tuberculosis H37Rv with LC3 in autophagosomes and an increase in phagolysosome fusion. We looked for the intracellular Mycobacterium tuberculosis H37Rv and H37Ra load in the cells by performing complimentary colony forming unit (CFU) (Figs. 6A and B) and LIVE/DEAD BacLight viability assays (Fig. 6C) on cells with and without GSK4112 alone or in combination with 3-MA. In keeping with an increase in autophagosomes, lysosomes, and phagolysosome fusion, GSK4112 induced clearance of Mycobacterium tuberculosis H37Rv. Similar results were observed in Mycobacterium tuberculosis H37Ra infected cells by CFU (Fig. 6B) and LIVE/DEAD BacLight viability assays (Fig. 6C). The GSK4112-induced clearance of Mycobacterium tuberculosis H37Rv was abrogated either in the presence of 3-MA or in conditions of BECN1 knockdown (Figs. 6A and D). The increased number of both autophagosomes and lysosomes indicates that the antimycobacterial role of NR1D1 is due to the enhancement of autophagy in addition to the repression of IL10. A schematic is drawn that depicts the NR1D1 mediated clearance of Mycobacterium tuberculosis clearance through modulation of autophagy and lysosome biogenesis (Fig. 6E).

Figure 5.

NR1D1 activation by its agonist enhances the numbers of autophagosomes and lysosomes. Enumeration of (A) autophagosomes and (B) lysosomes/autolysosomes in Mycobacterium tuberculosis H37Rv-infected THP-1 macrophages treated with GSK4112 in combination with bafilomycin A₁ and immunostained with anti-LC3 or anti-LAMP1 followed by Texas Red-conjugated anti-rabbit or anti-mouse antibody, respectively. Scale bar: 10 μm. The number of Texas Red-LC3-II and LAMP1 puncta in each cell were counted in 100 cells in each group. Quantification of the LC3 puncta per cell is represented as the mean ± SD of indicated number of experiments. Quantification of H37Rv with LC3 or LAMP1 is represented by overlay coefficient values. Images are representative of 5 independent replicates. *, P < 0.05 as compared to control or as indicated.

Figure 6.

GSK4112 augments Mycobacterium tuberculosis H37Rv clearance from macrophages. GSK4112 addition resulted in an increase in the intracellular Mycobacterium tuberculosis H37Rv and H37Ra clearance, as seen by the number of CFUs (A and B) and the percentage of dead bacteria measured by flow cytometry for H37Ra (C). GSK4112 mediated Mycobacterium tuberculosis H37Rv clearance was abrogated in BECN1 knockdown conditions (D). CFU counts are plotted as the mean ± SD, and flow cytometry results are plotted as the median. Data are representative of 3 independent experiments with similar results (n= 3; *, P < 0.05 as compared to control or as indicated). (E) Schematic representation of NR1D1 mediated clearance of Mycobacterium tuberculosis through autophagy.

Discussion

Autophagy represents one of several essential mechanisms that regulate the antimicrobial properties of macrophages.⁴⁷ This study conclusively shows that NR1D1 is an antimicrobial therapeutic target by virtue of its being a positive modulator of autophagy and lysosome biogenesis together with its ability to repress the IL10 gene in human macrophages. Either overexpression of NR1D1 or treatment with GSK4112 leads to an increase in LC3-II levels in a concentration- and time-dependent manner as seen in immunoblots and imaging by confocal microscopy (Figs. 1 and 2). On the basis of given reports on the correlation of an increase in autophagy with lysosome number, we explored the effects of NR1D1 activity on these processes. NR1D1 clearly modulates both processes which are associated with TFEB, a major transcription factor that links autophagy and lysosome biogenesis (Figs. 3, 4 and 5). We found that both LAMP1 protein levels, a marker of late endosomes-lysosome, and TFEB expression are significantly reduced in NR1D1 knockdown cells (Fig. 4). We also investigated the expression of the markers of autophagy and lysosome biogenesis along with the genes critical for the regulation of both pathways by real-time PCR (Fig. 4). Our results demonstrate that NR1D1 induced innate defense and autophagic flux in conjugation with a positive regulation on lysosome biogenesis together augment the clearance of Mycobacterium tuberculosis from human macrophages. This further strengthens the antimicrobial activity of NR1D1 and makes it a possible target for therapeutic application against Mycobacterium tuberculosis.

Autophagy is an intrinsic process ubiquitous to cells that has been studied under several physiological and pathological conditions.⁴⁸ It plays a major role as a defense mechanism against many intracellular pathogens, particularly Mycobacterium tuberculosis.^49,50 Previous studies have reported that the lysosomal system is also a notably effective barrier against many intracellular pathogens including Mycobacterium tuberculosis.^51,52 By virtue of regulating both these antimicrobial processes, NR1D1 could be a potential therapeutic target. However, the coevolution of both the host system and the pathogen has often allowed the pathogen to circumvent this defense mechanism. In Mycobacterium tuberculosis infection, the bacterium blocks the maturation and biogenesis of phagolysosomes, and thus the autophagosomes containing bacteria do not accomplish the late endosomal fusion with lysosomes.^53,54 In our earlier study, we demonstrated direct gene repression of IL10 by NR1D1, which abrogates this phagolysosome-maturation block.¹⁰ In response to several conditions such as environmental stress, infection and inflammation, various cytokines modulate both autophagy and lysosome biogenesis.^55,56 Cytokines, such as, IFNG, TNF, IL1, and IL2, have been shown to promote autophagy; whereas, IL4, IL10, and IL13 are inhibitory.^57,58 IL10, one of the major immunosuppressants, blocks the release of proinflammatory cytokines and inhibits phagolysosome maturation.⁵⁹ It also inhibits the microbicidal activity of macrophages by blocking a costimulatory factor, TNF, required for IFNG-induced activation.⁶⁰ In murine macrophages, IL10 activates the PI3K-AKT pathway and thereby inhibits starvation-induced autophagy.⁵⁸ It has also been reported to impede the clearance of several intracellular pathogens such as Mycobacterium tuberculosis, Listeria monocytogens, and Plasmodium sp.⁶¹ Consequently, IL10 and its regulation in infection and inflammation have always been considered intriguing for therapeutic manipulations.

In the past decade, several functions of NR1D1 have been studied, ranging from cell differentiation to metabolism.^4,62 NR1D1 has been termed a gatekeeper because of its timely coordination of metabolic responses.⁴ More recently, its antimicrobial activity has also been studied.¹⁰ Recently, Woldt, et al, 2013, have determined that in skeletal muscle cells NR1D1 controls mitochondrial biogenesis and respiration through the LKB1-AMPK-SIRT1-PPARGC1A signaling pathway in addition to regulating expression of genes specific for mitophagy.³⁹ In our study on macrophages, NR1D1 modulates lysosome biogenesis by positive regulation of TFEB expression, a master transcription regulator that coordinately regulates both autophagy and lysosome biogenesis. As mentioned above, knockdown of NR1D1 results in a decrease in expression of TFEB. Since NR1D1 is a repressor and the mechanism by which its knockdown modulates TFEB is not clear, there are 2 schools of thought about the mechanism. One possibility is that NR1D1 is negatively regulating a repressor of TFEB; the second is that it exerts a positive response on TFEB gene regulation. The later hypothesis is very recent; for the first time NR1D1 has been reported to activate transcription of the GJA1/connexin 43 gene by forming a complex with SP1 for this transactivation; moreover, ligand is dispensable.^63,64 Further investigation is required to determine which among these theories is relevant in this context. In skeletal muscle cells NR1D1 promotes mitochondrial biogenesis in addition to mitophagy abrogation; however, in macrophages it promotes lysosome biogenesis along with macroautophagy. This could possibly be because of the difference in cell type and function of NR1D1 in these cells. In skeletal muscle cells, more mitochondrial biogenesis is required to improve the oxidative capacity of the cells. On the other hand, more lysosome biogenesis is essential to augment the antimicrobial properties of macrophages.

Lastly, we have also addressed the important antimicrobial activity in human macrophages of the ligand of NR1D1, GSK4112. GSK4112 has been shown to regulate NR1D1 repressive activity in hepatocytes and macrophages by recruitment of NCOR and HDAC3 corepressors, consequently reducing the expression of genes involved in circadian biology, metabolism, and inflammation.⁶⁵ Thus, GSK4112 could be considered for use as an anti-infective compound against intracellular pathogens.

Materials and Methods

Reagents

Reagents were purchased from the indicated companies: Monodansylcadaverine (Sigma Aldrich, D4008), acridine orange (Sigma Aldrich, A6014), 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI; Sigma Aldrich, D8417), rabbit polyclonal antibody against LC3 (Sigma Aldrich, L8918), bafilomycin A₁ (Sigma Aldrich, B1793), 3-methyladenine (Sigma Aldrich, M9281), phorbol 12-myristate 13-acetate (Calbiochem, 524400), DMSO (Sigma Aldrich, D8418), SR8278 (Sigma Aldrich, S9576). LysoTracker Red (Life Technologies, L-7528) and antifade reagent (Life Technologies, P36934). GSK4112 (Tocris Biosciences, 3663). Horseradish peroxidase conjugated anti-mouse (Santa Cruz Biotechnology, sc-2314) and anti-rabbit (Santa Cruz Biotechnology, sc-2313), Texas Red conjugated anti-mouse IgG (Santa Cruz Biotechnology, sc-2781) and anti-rabbit IgG (Santa Cruz Biotechnology, sc-2780) and FITC conjugated anti-rabbit IgG (Santa Cruz Biotechnology, sc-2012).

Cell culture and Mycobacterium tuberculosis infection

THP-1 (NCCS, India) cells were maintained in RPMI-1640 medium (Gibco, Life Technologies, 11875) supplemented with 10% fetal bovine serum (Gibco, Life Technologies, 26140079), 100 U/ml penicillin, and 100 µg/ml streptomycin (Gibco, Life Technologies, 10378-016) in a 5% CO₂ incubator at 37°C. Cells were differentiated into macrophages by PMA treatment at a concentration of 30 ng/ml for 12 h followed by a 24-h resting period. pSC301 GFP vector capable of expressing in Mycobacterium sp and Escherichia coli was kindly provided by Dr. Yossef Av-Gay (University of British Columbia, Vancouver, BC, Canada). GFP-H37Ra was made by electroporation and selection as described previously.⁶⁶ Mycobacterium tuberculosis H37Rv and H37Rv-GFP culture were grown in Middlebrook 7H9 medium (BD Difco Laboratories, 271310) supplemented with 0.2% glycerol (Sigma Aldrich, G2025), 0.05% Tween 80 (Sigma Aldrich, P4780) and 10% Middlebrook oleic acid albumin dextrose catalase (OADC; BD Difco Laboratories, 211886) to log phase. Cultures of log phase were used for infection. Differentiated macrophages were infected with Mycobacterium tuberculosis strain H37Rv or H37Rv-GFP at multiplicity of infection 1:5 for 4 h, washed thrice with medium to remove unphagocytosed bacteria, and incubated with repletion medium for 24 h.

Plasmids and transfection

pCMV2-Flag-NR1D1 construct was prepared as described previously.¹⁰ siRNA was transfected into the cells by using Lipofectamine RNAiMAX reagent (Invitrogen, 13778-150) and plasmids were transfected using Lipofectmine Plus reagent, according to manufacturer's instructions (Invitrogen, 15338-100). The siRNA pair was selected on the basis of its binding to the 5′-UTR of the gene with no off targets.

Acridine orange, MDC and LysoTracker Red staining

THP-1 macrophage cells were treated with GSK4112 for 12 h followed by incubation with 0.05 mM MDC or 1 µg/ml AO for 15 min. Cells were then fixed with 4% paraformaldehyde (PFA) at room temperature and immediately observed under a Carl Zeiss Axioplan immunofluorescence microscope (Zeiss, Oberkochem, Germany) (at CSIR-IMTECH, India). For LysoTracker Red staining, cells were incubated with 100 nM LysoTracker Red for 30 min at 37°C followed by fixation with 4% PFA. Cells were then observed either with a flow cytometer (BD FACSVerse, BD Biosciences, California, USA) or with a confocal microscope (Nikon A1R, Nikon, Yokohama, Japan) at CSIR-IMTECH, India.

Real-time quantitative RT-PCR (qPCR)

Total RNA was isolated by the Trizol method from macrophages and monocytes; 1 µg was reverse transcribed (Fermentas, K1622) according to the manufacturer's protocol and subsequently amplified by PCR using specific primers. RNA18S/18S rRNA was also amplified in the samples and used as a loading control. The relative fold change of the target gene was calculated by the formula 2^−ΔΔCT, where ΔC_T is the difference between the values for the target gene and RNA18S rRNA. All experiments were conducted in triplicate.

Western blot analysis

For immunoblotting, 40 µg of total protein extract were resolved by SDS-PAGE on a 10% acrylamide gel (Bio-Rad) and transferred to a PVDF membrane. ACTB (Santa Cruz Biotechnology, sc-47778) were used as loading controls for the whole cell extract. The antigen-antibody (Ag-Ab) complexes were labeled with the appropriate HRP-conjugated secondary Abs and visualized by Immobilon western Chemiluminescent HRP Substrate (GE Healthcare, RPN2209; Milipore, WBLUF0500).

Confocal microscopy

After the relevant treatments, THP-1 macrophage cells were fixed with 4% PFA and processed for immunostaining. Intracellular staining of LC3 was performed using rabbit-anti-LC3 followed by FITC or Texas Red conjugated with goat-anti-rabbit antibody; LAMP1 was stained using mouse-anti-LAMP1 followed by Texas Red conjugated with goat-anti-mouse antibody. DAPI was used as a nuclear stain. For colocalization of mycobacteria with autophagososmes, GFP-H37Rv-infected macrophage cells were incubated for another 24 h. The coverslips were washed with phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl, 8 mM Na₂HPO₄, 1.47 mM KH₂PO₄, pH 7.4) and mounted on slides with antifade reagent. The stained cells were observed with a Nikon A1R confocal microscope. Cells with 10 or more LC3 puncta were scored as LC3-positive cells. Puncta formation was counted for at least 100 cells for each treatment and plotted as percentage positive cells with fluorescent LC3 or LAMP1 puncta. The colocalization of LC3, LAMP1 and GFP-H37Rv was quantified by selecting a region of interest and determining the overlap coefficient.⁶⁷

Mycobacterial viability determination by flow cytometry and CFU assay

Infected macrophage cells were lysed with 0.06% SDS (Sigma Aldrich, L3771) after 24 h of incubation, and bacterial suspensions were used with the LIVE/DEAD BacLight Bacterial Viability and Counting kit as per the manufacturer's instructions (Invitrogen, L34856). The percentage of live and dead bacteria was determined by flow cytometry (BD FACSAccuri, BD Biosciences, California, USA) after staining with SYTO9 and propidium iodide (PI) (LIVE/DEAD® BacLight™). For CFU determination after macrophage solubilization, the bacterial suspensions were serially diluted, 50 µl of each dilution was plated, and CFU were counted. Final calculations included the dilution factor and the volume of diluted sample used for plating.

Statistics

Statistical significance was determined using paired 2-tailed Student t test calculated using the Prism software. The results are expressed as mean ± standard deviation unless otherwise mentioned.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Supplemental Material

Supplemental data for this article can be accessed on the publisher's website.

Funding

This work was supported by Department of Biotechnology-India; project BT/01/IYBA/2009. This work was supported by the Department of Biotechnology-India project BT/01/IYBA/2009 and CSIR 12th Plan Network project Bugs to Drugs, Infectious Disease (BSC0211, BSC0210) to PG. We thank the Council of Scientific and Industrial research-Institute of Microbial Technology (CSIR-IMTECH), for facilities and financial support.