Glia Maturation Factor-γ Negatively Modulates TLR4 Signaling by Facilitating TLR4 Endocytic Trafficking in

This information is current as Wulin Aerbajinai, Kevin Lee, Kyung Chin and Griffin P. of September 28, 2021. Rodgers J Immunol 2013; 190:6093-6103; Prepublished online 15 May 2013; doi: 10.4049/jimmunol.1203048

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Glia Maturation Factor-g Negatively Modulates TLR4 Signaling by Facilitating TLR4 Endocytic Trafficking in Macrophages

Wulin Aerbajinai, Kevin Lee, Kyung Chin, and Griffin P. Rodgers

TLR4 signaling must be tightly regulated to provide both effective immune protection and avoid inflammation-induced pathology. Thus, the mechanisms that negatively regulate the TLR4-triggered inflammatory response are of particular importance. Glia mat- uration factor-g (GMFG), a novel depolymerization factor/cofilin superfamily that is expressed in inflammatory cells, has been implicated in mediating neutrophil and T , but its function in immune response remains unclear. In the current study, the role of GMFG in the LPS-induced TLR4-signaling pathway was investigated in THP-1 macro- phages and human primary macrophages. LPS stimulation of macrophages decreased GMFG mRNA and protein expression. We Downloaded from show that GMFG negatively regulates LPS-induced activation of NF-kB–, MAPK-, and IRF3-signaling pathways and subsequent production of proinflammatory cytokines and type I IFN in human macrophages. We found that endogenous GMFG localized within early and late endosomes. GMFG knockdown delayed LPS-induced TLR4 internalization and caused prolonged TLR4 retention at the early endosome, suggesting that TLR4 transport from early to late endosomes is interrupted, which may contribute to enhanced LPS-induced TLR4 signaling. Taken together, our findings suggest that GMFG functions as a negative regulator of TLR4 signaling by facilitating TLR4 endocytic trafficking in macrophages. The Journal of Immunology, 2013, 190: 6093–6103. http://www.jimmunol.org/

oll-like receptors, which are broadly distributed on the LPS, into the early/sorting endosome network, where the second cells of the immune system, play a critical role in innate signaling pathway is triggered through TRIF and TRIF-related T and adaptive immune responses through recognition of adaptor molecule (9–11). TLR4 is then either sorted to late pathogenic molecules and triggering of an inflammatory response endosomes and lysosomes for its degradation or sent to recycling (1, 2). TLR4 is the most extensively studied TLR in both innate compartments to be returned to the plasma membrane. Accord- immunity and signal transduction. It senses LPS from Gram- ingly, LPS-induced of TLR4 is essential for its sig- negative bacteria and initiates two separate signaling pathways naling function. Recent studies revealed that Rab family members through the recruitment of MyD88 or TRIF, which eventually regulate TLR4 trafficking. For example, Rab7b localizes to the by guest on September 28, 2021 induces the production of proinflammatory cytokines and type I late endosomes, where it negatively regulates TLR4 trafficking to IFN by activation of NF-kB, MAPK, and IRF3 (3, 4). LPS- late endosomes for degradation (12). Rab10 localizes to both the induced TLR4 signaling is tightly controlled to prevent exces- Golgi and early endosomes, where it regulates TLR4 signaling by sive inflammation and to allow for tissue repair and the return to promoting transport of TLR4 from the Golgi to the plasma mem- homeostasis postinfection. Uncontrolled or prolonged activation brane (13). Rab11a is enriched in pericentriolar-recycling endo- of TLR4 can have devastating consequences, including the de- somes and is essential for the trafficking of TLR4 into the Rab11a+ velopment of immunopathological conditions, such as autoim- endocytic recycling compartment (14). Although these GTPase mune diseases and lethal Gram-negative septic shock (5, 6). Rabs facilitate vesicle intracellular trafficking, little is known about Accordingly, much attention has focused on investigating how the surrounding cytoskeletal that regulate endosomal traf- to negatively regulate TLR4-signaling pathways (7). Although ex- ficking and the degradation pathway of the TLR4 receptor. tensive studies revealed that the negative regulation of TLR4 Glia maturation factor-g (GMFG), a newly recognized actin signaling occurs through various mechanisms (8), regulation of depolymerization factor/cofilin superfamily protein, is not in- the fine-tuning of TLR4-induced signaling by endocytosis and the volved in the development of glia or the formation of gliomas; factors that restrict these processes remain largely unclear. rather, it is predominantly expressed in inflammatory cells and Endocytic trafficking in the TLR4-signaling pathway recently microendothelial cells (15). GMF is a highly conserved protein emerged as a key feature of TLR4 function. Upon LPS stimulation, throughout the eukaryotes from yeast to mammals, with two TLR4 initiates the first MyD88-dependent signaling pathway at isoforms being expressed in mammals: GMFB and GMFG. Our the plasma membrane and is subsequently internalized, along with previous study (16) and other investigators (17) suggested that GMFG mediates neutrophil and T lymphocyte migration via regu- Molecular and Clinical Hematology Branch, National Heart, Lung, and Blood Insti- lation of actin cytoskeletal reorganization. Functional studies of tute, National Institutes of Health, Bethesda, MD 20892 Saccharomyces cerevisiae showed that yeast GMF promotes Received for publication November 2, 2012. Accepted for publication April 9, 2013. debranching of actin filaments and inhibits new actin assembly This work was supported by the Intramural Research Program of the National Insti- through binding of the Arp2/3 complex (18). Recent studies dem- tute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health. onstrated that the Arp2/3 complex and its regulating factors are Address correspondence and reprint requests to Dr. Griffin P. Rodgers, Molecular and involved in endosomal dynamics, as well as intracellular protein Clinical Hematology Branch, National Institutes of Health, Building 10, Room 9N- 113A, Bethesda, MD 20892. E-mail address: [email protected] trafficking (19, 20). Abbreviations used in this article: GMFG, glia maturation factor-g; Q-PCR, quanti- In the current study, we examined the role of GMFG in LPS- tative PCR; siRNA, small interfering RNA. initiated TLR4 signaling in human macrophages. We found that www.jimmunol.org/cgi/doi/10.4049/jimmunol.1203048 6094 GLIA MATURATION FACTOR-g NEGATIVELY REGULATES TLR4 SIGNALING

GMFG could serve as a negative regulator of TLR4 signaling in sitometric quantification of signals on blotting membranes was performed macrophages via promoting TLR4 receptor trafficking from early using ImageJ software (National Institutes of Health). endosomes to late endosomes. Flow cytometry analysis To detect cell surface expression of TLR4, PMA-differentiated THP-1 Materials and Methods macrophages were treated or not with 100 ng/ml LPS for 1 h, dissoci- Cells and cultures ated, blocked in 5% BSA for 30 min, stained with FITC-conjugated anti- TLR4 (Imgenex) or IgG2a Ab for 30 min on ice, washed, and analyzed by The human monocytic leukemia cell line THP-1 was obtained from the EPICS ELITE ESP flow cytometry (Beckman Coulter); the data were American Type Culture Collection and cultured in RPMI 1640 with 10% analyzed using CellQuest software (BD Biosciences). Cell surface ex- heat-inactivated FBS, 50 mM 2-ME, and 1% penicillin/streptomycin at pression of TLR4 was quantified as the mean fluorescence intensity of the 37˚C in a humidified atmosphere, consisting of 5% CO ,andpassed 2 FITC+ cells by FACS assay. every 2–3 d to maintain logarithmic growth. THP-1–derived macrophages 5 were obtained by stimulating THP-1 monocytes (5 3 10 /ml) with 100 ng/ Immunofluorescence staining and confocal microscopy ml PMA (Sigma-Aldrich) for 3 d. Human monocytes were isolated from healthy adult donors according to THP-1 macrophages and primary human macrophages were transfected a protocol approved by the Institutional Review Board of the National Heart, with control siRNA or GMFG siRNA for 48 h. The cells were then treated Lung, and Blood Institute, National Institutes for Health, and consistent with 100 ng/ml LPS for the indicated time periods. Following treatment, with federal regulations. Monocytes were isolated by Ficoll-Paque (GE cells were fixed in 4% paraformaldehyde/PBS for 20 min, permeabilized Healthcare) density centrifugation and magnetically sorted using CD14+ with 0.1% Triton X-100/PBS for 10 min, and preblocked in 10% FBS/PBS magnetic beads (MACS; Miltenyi Biotec), according to the manufacturer’s for 1 h. For TLR4 staining, after fixation with 4% paraformaldehyde, cells instructions. Isolated monocytes were counted and were 85–95% CD14+ were fixed with ice-cold methanol for 20 min. Cell were then stained with cells. Primary human macrophages were generated by differentiation of mouse monoclonal anti-TLR4 Ab (Abcam; 1:100) for 3 h, followed by Downloaded from CD14+ monocytes in complete culture medium (RPMI 1640 with 10% staining with rabbit anti-Rab5, anti-Rab4, or anti-Rab7 ( human AB serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 mg/ml Technology) for 1 h. After washes, the cells were stained with secondary streptomycin) supplemented with 10 ng/ml recombinant human GM-CSF Alexa Fluor 488 anti-mouse or Alexa Fluor 594 anti-rabbit Ab (Invitrogen), (R&D Systems). The medium was changed 3 d after seeding, and cells diluted 1:500, for 1 h at room temperature. Nuclear DNA was stained with developed the macrophage phenotype after 7 d in culture. DAPI (Sigma) for 5 min. For the colocalization analysis of GMFG and organelle markers, cells Isolation of RNA and quantitative PCR were sequentially immunostained with GMFG Ab overnight at 4˚C and stained with Ab against EEA1, Rab5, Rab7, Golgi marker GM130 (BD http://www.jimmunol.org/ Total RNA was prepared using the RNeasy Plus Mini kit (QIAGEN), Pharmingen), or endoplasmic reticulum marker PDI (Invitrogen), followed according to the manufacturer’s instructions. For reverse transcription, by staining with the appropriate Alexa Fluor 488–conjugated or Alexa 1 mg total RNA/sample was used as a template for cDNA synthesis using Fluor 594–conjugated secondary Ab (Invitrogen). Cells were examined Superscript III (Invitrogen), following the manufacturer’s guidelines. under a Zeiss LSM510 META confocal microscope equipped with 405-, Quantitative PCR (Q-PCR) reactions (MyiQ iCycler; Bio-Rad) were per- 488-, 594-, and 633-nm lasers and Zen 2009 imaging software, using formed using prevalidated TaqMan primer/probe sets purchased as Assays- a633/1.3 NA oil-immersion objective (Carl Zeiss). Quantitative coloc- on-Demand -expression products from Applied Biosystems. Real- alization analysis was performed using Imaris 7.6 software (Bitplane). time PCR conditions were 5 min at 95˚C and 40 cycles of 30 s at 95˚C, Dual-color scattergrams and the percentage of colocalization were mea- followed by 1 min at 60˚C. RNA copy numbers were calculated by sured using the Pearson coefficient to evaluate the overlap in fluorescence comparison to standard curves generated from plasmid DNA–encoding

of TLR4 or GMFG with other endosome markers from four images taken by guest on September 28, 2021 GMFG and control b-actin templates. from two independent experiments. RNA interference and plasmid constructions Subcellular fractionation Day 1–differentiated THP-1 macrophages (2 3 106 cells) or primary hu- For subcellular fractionation, THP-1 macrophages were placed on ice, man macrophages (2 3 106 cells) were transiently transfected with rinsed twice with ice-cold PBS, and subjected to Percoll density-gradient a GMFG small interfering RNA (siRNA), negative-control siRNA, GMFG- cell fractionation and centrifugation, which were performed as previ- GFP plasmid, or GFP empty vector, using the Nucleofector Kit V and ously described (12). A total of 12 fractions was collected, starting from Nucleofector I Program U-01 (Amaxa Biosystems), according to the the top of the gradient. For immunoblotting analysis of each fraction, equal manufacturer’s protocol. GMFG siRNA (Silencer Select siRNA s18303) aliquots from each fraction were resolved by SDS-PAGE and then trans- and negative-control siRNA (Neg-siRNA #2) were obtained from Applied ferred to nitrocellulose, according to standard techniques. Biosystems. The cells were continually cultured in complete RPMI 1640 medium containing 10% FBS and 100 ng/ml PMA for 48 h prior to use. Statistical analysis Cytokine assays The Student t test was used to analyze data for significant differences. The p values , 0.05 were regarded as statistically significant. THP-1 macrophages were stimulated with 100 ng/ml LPS for the indicated time periods. The levels of TNF-a, IL-6, IFN-b, and IL-10 in culture supernatants were measured using commercial ELISA kits from BD Results Biosciences, according to the manufacturer’s instructions. GMFG is downregulated by LPS stimulation in macrophages Luciferase reporter assay GMFG is expressed mainly in inflammatory cells and micro- The determinations of NF-kB activity were performed as previously de- endothelial cells, but its biological function, especially in the innate scribed (21, 22). immune response in macrophages, remains unclear. To explore the possible functions of GMFG in the TLR4-signaling pathway, we Immunoblotting analysis first investigated whether GMFG could be regulated by LPS in THP-1 macrophages and primary human macrophages were lysed in RIPA human macrophages. In primary human macrophages, we found protein extraction reagent (Pierce Biotechnology), as recommended by the a substantial decrease in expression of GMFG mRNA and protein manufacturer. Equal amounts of total protein (40 mg/lane) were separated on 4–20% SDS-polyacrylamide gels and transferred to nitrocellulose after stimulation with 100 ng/ml LPS for 4 h that remained low over membranes using standard methods. Membranes were probed with pri- 24 h (Fig. 1A). The inhibitory effect at 4 h was concentration de- mary Abs for GMFG (ABGENT), TLR4 (Abcam), b-actin, phosphory- pendent and was evident at a range of LPS concentrations from 100 lated forms of Erk1/2 (Thr202/Tyr204), p38 (Thr180/Tyr182), JNK1/2 to 1000 ng/ml (Fig. 1B). We further verified this effect in THP-1 (Thr183/Tyr185), IRF3 (Ser396), NF-kB p65, and IkBa (Ser32/36), total p38, NF-kB, or IkBa (Cell Signaling Technology). HRP-conjugated anti- macrophages produced through PMA stimulation of THP-1 mono- mouse, -rabbit, or -goat IgG secondary Abs and the Amersham ECL cytes. Similarly, expression of GMFG mRNA and protein decreased Western blotting system (GE Healthcare) were used for detection. Den- in these cells upon stimulation with LPS (Fig. 1C). These results The Journal of Immunology 6095 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 1. Silencing of GMFG expression enhances LPS-induced production of proinflammatory mediators in macrophages. (A–C) GMFG expression upon LPS stimulation. Primary human macrophages (A, B) and THP-1 macrophages (C) were treated with 100 ng/ml LPS for the indicated times or with the indicated doses of LPS for 24 h; GMFG mRNA expression levels were assessed by Q-PCR, and protein levels were determined by immunoblotting. b-actin was used as an internal control. (D and E) Primary human macrophages and THP-1 macrophages were transfected with control negative siRNA (Ctrl siRNA) or GMFG siRNA for 48 h. The efficiency of knockdown was evaluated by Q-PCR and immunoblotting analysis. (F and G) Primary human macrophages transfected with Ctrl siRNA or GMFG siRNA were treated with 100 ng/ml LPS for the indicated times. TNF-a, IL-6, IFN-b, and IL-10 mRNA expression levels were assessed by Q-PCR, and cytokine production was measured by ELISA. Data are mean 6 SD of three independent experiments. *p , 0.05, **p , 0.01. suggest that GMFG may be involved in regulation of TLR4 sig- the production of TNF-a, IL-6, and IFN-b in response to LPS naling in macrophages. stimulation at both the mRNA and protein level compared with control siRNA–transfected primary human macrophages (Fig. 1F, Silencing of GMFG enhances the production of LPS-induced proinflammatory mediators in macrophages 1G). In contrast, IL-10 mRNA and protein levels were decreased in response to LPS in GMFG-knockdown macrophages compared To determine whether the downregulation of GMFG expression with control siRNA–transfected cells. Similar changes were ob- following LPS stimulation has any functional consequences, we served in THP-1 macrophages (data not shown). Because the LPS- silenced the expression of GMFG in human macrophages using a a previously verified GMFG siRNA (16). The knockdown effi- induced production of TNF- and IL-6 is related to LPS-initiated ciency and specificity of the siRNA oligonucleotides were con- MyD88-mediated NF-kB or MAPK activation, whereas the LPS- firmed to be .70% in primary human macrophages and THP-1 induced IFN-b production is largely dependent on TRIF-mediated macrophages (Fig. 1D, 1E). The effect of GMFG knockdown on IRF3 activation, these data suggest that GMFG mediates regula- LPS-induced inflammatory responses was then examined in hu- tion of the innate immune response in macrophages via MyD88- man macrophages. Knocking down GMFG significantly enhanced dependent and TRIF-dependent TLR4-signaling pathways. 6096 GLIA MATURATION FACTOR-g NEGATIVELY REGULATES TLR4 SIGNALING Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 2. Silencing of GMFG expression potentiates the LPS-induced signaling pathway in macrophages. THP-1 macrophages and primary human macrophages were transfected with control negative siRNA (Ctrl siRNA) or GMFG siRNA. After 48 h, the cells were treated with 100 ng/ml LPS for the indicated times. Cell lysates extracted from THP-1 macrophages (A) or primary human macrophages (B) were prepared and subjected to immunoblotting with the indicated Abs. NF-kB was used as an internal control. Relative quantification of phosphorylation of p65 and degradation of IkBa normalized to total NF-kB is shown in the lower panels. Data are mean 6 SD of three independent experiments. Values for control negative siRNA (Ctrl siRNA)– transfected cells not stimulated with LPS were set to 1. (C) Nuclear translocation of p65 was examined by confocal microscopy after LPS stimulation in GMFG siRNA–transfected or control negative siRNA (Ctrl siRNA)–transfected THP-1 macrophages. Cells were immunostained with anti–NF-kB p65 Ab followed by Alexa Fluor 488–conjugated secondary Ab. Scale bar, 50 mm. Quantification of p65 translocation to the nuclei (right panel). Data are mean 6 SD of three coverslips from a representative experiment. A total of 100 cells was counted per coverslip. (D) THP-1 macrophages that had been knocked down with GMFG siRNA or control negative siRNA were transfected with pTK-Renilla-luciferase and NF-kB luciferase reporter plasmids. After 24 h, the cells were stimulated with 100 ng/ml LPS for 6 h, and NF-kB luciferase activity was measured using the Dual-luciferase Reporter Assay System (Promega), normalized to the internal control Renilla luciferase activity. Values for mock-transfected cells not stimulated with LPS were set (Figure legend continues) The Journal of Immunology 6097 Downloaded from http://www.jimmunol.org/ FIGURE 3. Overexpression of GMFG inhibits LPS-induced cytokine production and LPS-initiated signaling pathways in macrophages. THP-1 mac- rophages were transfected with GMFG-GFP plasmid or GFP empty vector. After 24 h, the cells were stimulated with 100 ng/ml LPS for the indicated times. (A and B) TNF-a, IL-6, IFN-b, and IL-10 mRNA expression levels were measured by Q-PCR, and cytokine production was measured by ELISA. Data are mean 6 SD of three independent experiments. (C) Cell lysates from GMFG-GFP–transfected and GFP vector–transfected THP-1 macrophages were prepared and subjected to immunoblotting with the indicated Abs. b-actin was used as an internal control. *p , 0.05 versus control.

Silencing of GMFG expression potentiates LPS-initiated macrophages revealed remarkably enhanced LPS-induced phos- signaling pathways in macrophages phorylation of Erk1/2 compared with control siRNA–transfected

To investigate the molecular basis of the enhanced cytokine pro- cells, whereas LPS-induced phosphorylation of p38 and JNK was by guest on September 28, 2021 duction associated with GMFG knockdown, we subsequently not considerably increased in GMFG-silenced cells compared assessed the effect of GMFG knockdown on downstream-signaling with control-transfected cells (Fig. 2E, 2F). Finally, analysis of components of the TLR4-signaling pathway. We first evaluated the LPS-induced activation of IRF3 also showed increased phos- effect of GMFG knockdown on activation of NF-kB in THP-1 phorylation of IRF3 in GMFG-knockdown THP-1 macrophages macrophages and primary human macrophages. Knockdown of and primary human macrophages compared with control-transfected GMFG led to greatly enhanced phosphorylation of NF-kB p65 cells (Fig. 2E, 2F). Collectively, these results indicate that GMFG and degradation of IkBa after LPS treatment for 60 min in THP-1 may negatively regulate LPS-initiated activation of the NF-kB, macrophages and 30 min in primary human macrophages; this MAPK, and IRF3 pathways. effect was sustained for 6 h in THP-1 macrophages and for 1 h in primary human macrophages (Fig. 2A, 2B). In parallel with this Overexpression of GMFG inhibits LPS-induced cytokine immunoblotting analysis, fluorescence microscopy demonstrated production and LPS-initiated signaling pathways in enhanced NF-kB activation in GMFG-knockdown THP-1 mac- macrophages rophages. Pronouncedly enhanced nuclear import of p65 was To further support our findings that GMFG may negatively regulate observed as early as 5 min after LPS treatment of THP-1 mac- the LPS-initiated signaling pathway, we overexpressed GMFG- rophages, indicating nuclear translocation of the NF-kB p65 GFP in THP-1 macrophages. GMFG-overexpressing THP-1 subunit (Fig. 2C). Further, NF-kB luciferase reporter assays of macrophages produced significantly less TNF-a, IL-6, and IFN- THP-1 macrophages revealed that knockdown of GMFG en- b, but not IL-10, than did control-transfected cells in response hanced LPS-induced transcriptional activity of NF-kB promoters to LPS (Fig. 3A, 3B), indicating that GMFG overexpression in- compared with activity observed in control siRNA–transfected hibited both MyD88- and TRIF-dependent TLR4 signaling in re- cells (Fig. 2D). Next, we examined MAPK activation status after sponse to LPS in macrophages. We next evaluated the effect of LPS stimulation in GMFG-knockdown THP-1 macrophages and GMFG overexpression on the LPS-induced phosphorylation state primary human macrophages, because activation of the MAPK of NF-kB, Erk1/2, and IRF3 in THP-1 macrophages. We found pathways also plays an essential role in response to LPS. that GMFG overexpression could inhibit LPS-induced phosphor- Knockdown of GMFG in THP-1 macrophages and primary human ylation of Erk1/2 and IRF3 compared with control vector–trans-

to 1. Data are mean 6 SD of three experiments. Cell lysates extracted from GMFG siRNA–transfected or control negative siRNA (Ctrl siRNA)–transfected THP-1 macrophages (E) or primary human macrophages (F) were subjected to immunoblotting with the indicated Abs. Total p38 and b-actin were used as internal controls. Relative quantification of phosphorylation of Erk1/2 and IRF3 (lower panels). Data are mean 6 SD of three independent experiments. Values for control negative siRNA (Ctrl siRNA)–transfected cells not stimulated with LPS were set to 1. *p , 0.05, **p , 0.01. 6098 GLIA MATURATION FACTOR-g NEGATIVELY REGULATES TLR4 SIGNALING fected cells (Fig. 3C). We also noted that GMFG overexpression It is well established that activated TLR4 can be transported from had no effect on LPS-induced phosphorylation of p38 and JNK, the to endosomes for ubiquitination and to lyso- consistent with the effect of GMFG knockdown on p38 and JNK. somes for degradation (25). Thus, we examined, using confocal These data provide additional evidence that GMFG negatively microscopy, whether GMFG mediates subcellular trafficking of regulates TLR4-initiated signaling pathways. TLR4 after LPS stimulation. In unstimulated cells transfected with control siRNA, TLR4 was initially present at the cell surface Silencing of GMFG inhibits TLR4 internalization and in tiny intracellular vesicles that were distributed diffusely We next investigated the cellular mechanisms by which GMFG throughout the . After 10 min of LPS treatment, TLR4 could negatively regulate TLR4 signaling. Recent studies showed staining appeared increased in intracellular vesicles compared that regulation of TLR4 signaling is dependent on TLR4 expression with that in unstimulated cells, suggesting that TLR4 was inter- or TLR4 degradation (12, 23, 24). Thus, we determined whether nalized into the cytoplasm. At later time points (40 and 60 min) of GMFG could modulate the expression level of TLR4. Immuno- LPS stimulation, TLR4 had accumulated predominantly in the blotting analysis indicated that GMFG knockdown or overexpression perinuclear area (Fig. 4C, left panels). In contrast, in GMFG- did not affect total protein levels of TLR4 during LPS stimulation in knockdown cells, TLR4 staining appeared to be concentrated in THP-1 macrophages (Fig. 4A). However, flow cytometry analysis the periplasma membrane area at 10 min of LPS stimulation, and showed that GMFG knockdown slightly increased TLR4 surface it was sustained in the intracellular vesicles through to the late expression in both unstimulated and stimulated cells compared with time points (40 and 60 min) (Fig. 4C, right panels). In addition, control siRNA–transfected macrophages (Fig. 4B). These results control IgG or an isotype-matched (irrelevant) mAb showed suggested that GMFG effects on TLR4 signaling observed in our minimal (or no) background fluorescence for TLR4 staining (data Downloaded from earlier experiments likely were not attributable to changes in cell not shown). These results indicate that GMFG may be involved in surface or total protein expression levels of TLR4. LPS-induced TLR4 internalization. http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 4. Silencing of GMFG inhibits TLR4 internalization. (A) Immunoblotting analysis of TLR4 expression in GMFG-silenced or GMFG-over- expressed THP-1 macrophages. Cells were transfected with GMFG siRNA, control negative siRNA (Ctrl siRNA), GFP empty vector, or GMFG-GFP plasmids. After 48 h, the cells were treated with 100 ng/ml LPS for the indicated times, and cell lysates were prepared and subjected to immunoblotting with TLR4, GMFG, or GFP Ab. b-actin was used as an internal control. (B) Cell surface TLR4 expression was evaluated by flow cytometry using a specific anti-TLR4 Ab in THP-1 macrophages transfected with GMFG siRNA or control negative siRNA (Ctrl siRNA). Mean fluorescence intensity is shown. (C) TLR4 localization was examined by confocal microscopy after LPS stimulation in GMFG siRNA–transfected or Ctrl siRNA–transfected THP-1 macro- phages. Cells were immunostained with anti-TLR4 Ab, followed by Alexa Fluor 488–conjugated secondary Ab. Images are representative of three in- dependent experiments. Scale bar, 100 mm. The Journal of Immunology 6099

GMFG colocalizes with early and late endosome compartments Consistent with our confocal microscopy studies, we found that To determine the functional consequence of disrupting the LPS- GMFG was mainly localized in fractions positive for the early induced internalization of TLR4 by GMFG knockdown in THP- endosome marker EEA1 (fractions 1–3), with some GMFG present 1 macrophages, we examined the subcellular localization of en- in fractions positive for the late endosome marker Rab7. In ad- dogenous GMFG in THP-1 macrophages by confocal microscopy. dition, a partial overlap was observed between GMFG and the A large portion of the GMFG was displayed throughout the cy- lysosomal marker cathepsin D in fractions 3 and 12 (Fig. 5F). toplasm of the cells, with some high-intensity spots being readily Altogether, these results suggest that GMFG is an early and late apparent. These spots were reduced in intensity upon GMFG endosome–associated protein in THP-1 macrophages. knockdown, confirming the staining’s specificity (data not shown). Moreover, control IgG or an isotype-matched (irrelevant) mAb Silencing of GMFG induces abnormal TLR4 endosomal showed minimal (or no) background fluorescence for GMFG trafficking staining (data not shown). To characterize these spots, we attempted Given that GMFG colocalized with EEA1 and Rab5 on early/ to colocalize them with a series of well-characterized organelle sorting endosomes and with Rab7 on late endosomes, it is membrane markers. Costaining with different membrane-specific highly likely that GMFG may be involved in transport between markers revealed that endogenous GMFG was highly colocalized these endocytic compartments. Thus, we examined the effect with the early/sorting endosome markers EEA1 and Rab5 (Fig. 5A, of GMFG knockdown on TLR4 endocytic trafficking in LPS- 5B), as well as the late endosome marker Rab7 (Fig. 5C), but it was stimulated THP-1 macrophages and primary human macro- not colocalized with the Golgi marker GM130 (Fig. 5D) or the en- phages. GMFG knockdown delayed TLR4 trafficking to Rab5+ doplasmic reticulum marker PDI (Fig. 5E), suggesting direct linkage early endosomes at early time points (10 min) of LPS stimulation, Downloaded from of GMFG to the early and late endosomes in THP-1 macrophages. because we observed that TLR4 did not colocalize with Rab5+ We performed subcellular fractionation using Percoll density- endosomes at 10 min after LPS stimulation in GMFG siRNA– gradient centrifugation to confirm these steady-state findings. transfected cells, but it could be detected in early endosomes at 10 http://www.jimmunol.org/

FIGURE 5. GMFG colocalizes by guest on September 28, 2021 with early and late endosome com- partments. (A–E) Confocal microscopy of THP-1 macrophages immuno- stained with anti-GMFG Ab (green), along with Abs for the indicated organelle membrane markers (red). Scale bar, 100 mm. Dual-color pixel analysis of the colocalization of GMFG and various organelle markers (right panels). Data are mean 6 SD of three independent experiments. (F) Steady-state distribution of organelle marker proteins following subcellular fractionation using Percoll density- gradient centrifugation. Postnuclear supernatants of THP-1 macrophages were fractionated on 17% Percoll- density gradients, and the individual fractions were examined by immu- noblotting with specific early endo- some (EEA1), late endosome (Rab7), and lysosome (cathepsin D; Cell Signaling Technology) marker Abs, as well as GMFG Ab. 6100 GLIA MATURATION FACTOR-g NEGATIVELY REGULATES TLR4 SIGNALING min after LPS stimulation in control siRNA–transfected cells (Fig. staining accumulated in the perinuclear area, colocalized with late 6A). At late time points (40 and 60 min) of LPS stimulation in endosome marker Rab7 (Fig. 6B). GMFG-knockdown cells, TLR4 appeared to colocalize with To further determine whether GMFG is also involved in TLR4 Rab5+ endosomes (Fig. 6A), and substantially less TLR4 transport internalization, we examined TLR4 internalization upon LPS to Rab7+ late endosomes occurred (Fig. 6B). In control cells, stimulation from very early time points (5, 10, and 20 min) using following 40–60 min of LPS stimulation, TLR4 no longer colo- the fast recycling endosome marker Rab4 in GMFG-knockdown calized with Rab5+ early endosomes; instead, most of the TLR4 cells. Surprisingly, we found that TLR4 appeared to be colo- Downloaded from http://www.jimmunol.org/

FIGURE 6. Silencing of GMFG induces abnormal TLR4 endosomal trafficking. THP-1 macrophages and primary human macrophages were transfected with GMFG siRNA or control negative siRNA (Ctrl siRNA). After 48 h, the cells were stimulated or not with LPS for the indicated times. Following treatment, cells were by guest on September 28, 2021 fixed with 4% paraformaldehyde for 20 min and then in ice-cold methanol for another 20 min. Fixed THP-1 macrophages were immunostained with Abs against TLR4 (green) and Rab5 (early endosome marker; red) (A), Abs against TLR4 (green) and Rab7 (late endo- some marker; red) (B), or Abs against TLR4 (green) and Rab4 (fast recycling endosome marker; red) (C). Fixed primary human macrophages were immunos- tained with Abs against TLR4 (green) and Rab5 (red) (D) or Abs against TLR4 (green) and Rab7 (red) (E). Nuclear DNA was labeled with DAPI (blue). Scale bar, 100 mm. The Journal of Immunology 6101 calized with the Rab4+ compartment within 5 min of LPS stim- 100 ng/ml LPS for 24 h. Consistent with published reports de- ulation in control cells (Fig. 6C). Conversely, TLR4 transport to scribing LPS-induced endotoxin tolerance, pretreatment with low- Rab4+ endosomes at the very early time point (5 min) of LPS dose LPS nearly completely inhibited TNF-a, IL-6, and IFN-b stimulation appeared to be delayed in GMFG-knockdown cells, production after secondary LPS challenge in THP-1 macrophages but TLR4 appeared to be colocalized with the Rab4+ compartment transfected with control siRNA (Fig. 7A). GMFG-knockdown after 10 min of LPS stimulation (Fig. 6C). cells had substantially enhanced production of TNF-a, IL-6, and Finally, we examined the endocytic trafficking of TLR4 in IFN-b during the first 24 h after exposure to LPS in unprimed GMFG-knockdown primary human macrophages. Consistent with cells, whereas production of these cytokines was blocked over our data from THP-1 macrophages, GMFG-knockdown primary time in low-dose LPS-primed cells in response to secondary LPS human macrophages exhibited impaired TLR4 transport to the challenge (Fig. 7A). To determine whether pretreatment with Rab7+ late endosomes after 40 and 60 min of LPS stimulation LPS altered LPS responses at the level of , we (Fig. 6E) compared with control primary human macrophages. measured mRNA by real-time Q-PCR. Pretreatment with LPS Furthermore, in GMFG-knockdown primary human macrophages, substantially suppressed subsequent LPS-induced expression of TLR4 staining appeared to be retained in the cytoplasmic punc- mRNA for TNF-a, IL-6, and IFN-b in both control and GMFG- tate- and focal contact-like staining but not in the accumulated knockdown cells (Fig. 7B). These results indicate that GMFG is perinuclear area during the late time points of LPS stimulation not required for LPS-induced downregulation of cytokine pro- (Fig. 6D). However, we were unable to detect the colocalization of duction. TLR4 with the early endosome marker Rab5 in both control siRNA– and GMFG siRNA–transfected primary human macro- Discussion Downloaded from phages. This might represent a technical issue rather than a nega- Although extensive studies described the negative regulation of tive result. Taken together, these results indicate that GMFG not TLR4 signaling, and many negative regulators have been identified only mediates TLR4 transport from early to late endosomes, it (26–30), few reports exist about the regulators that control TLR4 also mediates TLR4 internalization. intracellular trafficking upon microbial recognition. In this study, we identified for the first time, to our knowledge, an important role GMFG is not required for endotoxin tolerance of GMFG in TLR4 signaling—that GMFG is a novel negative http://www.jimmunol.org/ Because GMFG knockdown enhanced the LPS-induced inflam- regulator of the MyD88-dependent and TRIF-dependent TLR4- matory response, we sought to investigate whether GMFG had signaling pathways—by demonstrating that GMFG mediated an effect on endotoxin tolerance in vitro. We pretreated THP-1 regulation of TLR4 endocytosis in macrophages. Our data reveal macrophages transfected with control siRNA or GMFG siRNA that depletion of GMFG results in abnormal trafficking of TLR4 in with 10 ng/ml LPS for 24 h and subsequently challenged them with endosomes in response to LPS stimulation. This abnormal accu- by guest on September 28, 2021

FIGURE 7. GMFG is not required for endotoxin tolerance. THP-1 macrophages were transfected with GMFG siRNA or control negative siRNA (Ctrl siRNA). After 48 h, the cells were treated with LPS (1st LPS; 10 ng/ml) for 24 h, the supernatants were removed, and the cells were washed and challenged with LPS (2nd LPS; 100 ng/ml) for another 24 h. (A) TNF-a, IL-6, and IFN-b levels were measured by ELISA. (B) TNF-a, IL-6, and IFN-b mRNA expression levels were assessed by Q-PCR. Data are mean 6 SD of three independent experiments. *p , 0.05, **p , 0.01. 6102 GLIA MATURATION FACTOR-g NEGATIVELY REGULATES TLR4 SIGNALING mulation of TLR4 in early endosomes may enhance the TLR4- significantly enhanced LPS-induced TLR4 signaling. Multiple signaling pathway. lines of evidence support this statement. First, we found that Endocytosis has emerged as a critical mechanism for controlling knockdown of GMFG enhanced LPS-induced phosphorylation of the signaling pathway via endosomal sorting of the internalized NF-kB, Erk1/2, and IRF3 and increased production of down- receptors, and their endocytic trafficking. Recent studies defined stream inflammatory cytokines, such as TNF-a, IL-6, and IFN-b. a requirement for actin dynamics in endocytosis in mammalian Second, although overexpression of GMFG did not inhibit LPS- cell lines (31, 32). Actin polymerization and depolymerization induced activation of NF-kB, it clearly inhibited LPS-induced are tightly regulated to guarantee the correct endocytic membrane Erk1/2 and IRF3 phosphorylation and downstream cytokines, transport. Several lines of evidence showed that actin-depolymerizing such as TNF-a, IL-6, and IFN-b. This hyperresponsiveness to LPS protein is involved in receptor trafficking from the endosome to under GMFG-knockdown conditions could be explained by pro- the recycling compartment and degradation in normal cells. For longed accumulation of TLR4 in the early endosomes. Third, LPS example, cofilin (actin depolymerization factor/cofilin superfamily induced downregulation of GMFG, which is consistent with member)-mediated actin filaments are required for the multiple a previous study finding in which transfection of EBV nuclear Ag steps of endocytic trafficking (33, 34). PICK1, an inhibitor of Arp2/ 2 in B cells also induced the downregulation of GMFG (47). This 3-mediated actin assembly, is required for AMPA receptor endo- finding suggested that LPS-induced GMFG downregulation is cytosis (35, 36). Therefore, regulation of actin patch disassembly required for inducing optimal activation of TLR4 signaling and plays an important role in endocytosis. GMFG, another actin de- may be the mechanism used by the host to avoid suppression of polymerization factor/cofilin superfamily protein, was reported to the innate immune response aimed at thoroughly removing in- function as an actin disassembly protein by inhibiting the Arp2/3 vading pathogenic microbes. Finally, GMFG is not required for Downloaded from complex in yeast (37). Although GMFG’s action as a disassembly/ LPS-induced endotoxin tolerance. GMFG is downregulated by debranching protein in mammalian cells has yet to be determined, it stimulation with LPS at doses of 100–1000 ng/ml. However, at has a particularly interesting role in TLR4 endocytosis. Our results lower doses of LPS stimulation (1–10 ng/ml), GMFG was not with GMFG are the first, to our knowledge, to characterize a role for downregulated. Therefore, our data suggest that regulation of GMFG in TLR4 trafficking in endosomes in response to LPS, as GMFG expression may not be a major mechanism by which the

evidenced by studies in GMFG-knockdown macrophages. cell regulates its responses to microbial stimuli. Given all of the http://www.jimmunol.org/ Our confocal microscopy study revealed that GMFG is found evidence obtained so far, our results strongly suggest that GMFG throughout the cytosol and colocalizes with the early and late may negatively regulate TLR4 signaling by the modulation of endosome compartments, indicating that GMFG may be involved TLR4 endocytic trafficking in human macrophages. in TLR4 endocytic trafficking. TLR4 endocytosis at the early phase In this study, our data showed that GMFG knockdown or of LPS stimulation is predominantly dependent (25, 38), overexpression did not affect total protein levels of TLR4, which whereas recent studies show that caveolae/lipid raft–mediated differs from previous studies in which total TLR4 expression is endocytosis and macropinocytosis also play important roles in increased along with accumulation of TLR4 in early endosomes TLR4 endocytosis and signaling activation (39–42). These endo- (12, 45). Although we cannot provide conclusive results for the cytosis pathways are maintained in a dynamic balance, and inhi- role of GMFG on TLR4 expression using the present data alone, by guest on September 28, 2021 bition of one endocytosis pathway can shift receptor trafficking to we have provided evidence that silencing of GMFG delayed TLR4 another pathway. For example, siRNA knockdown of clathrin internalization and induced abnormal accumulation of TLR4 in results in a significant inhibition of TLR4 uptake in the early phase early endosomes. These results suggest that activation of TLR4 of LPS stimulation, whereas TLR4 accumulates in the endosome at signaling after GMFG knockdown is a direct consequence of the late phase of LPS stimulation, indicating that TLR4 transport impaired intracellular membrane trafficking but is not due to occurs by a clathrin-independent pathway (25). Similarly, we ob- changing TLR4 expression levels. served that knockdown of GMFG inhibited TLR4 internalization to In summary, we identified and characterized a role for GMFG Rab4+ endosomes, but it appears to prolong the accumulation of as a negative regulator of TLR4 signaling, which is facilitated by TLR4 in Rab5+ early endosomes, which impairs TLR4 transloca- TLR4 endocytic trafficking that is evoked by its ligand LPS. tion to Rab7+ late endosomes for further degradation. Thus, it is Hence, further studies of the role for GMFG in the regulation of likely that knockdown of GMFG leads to prolonged TLR4 retention TLR family members are likely to identify useful therapeutic in the plasma membrane, which might enhance LPS-induced NF- targets for the treatment of patients with autoimmune, chronic kB activation. However, the mechanisms underlying GMFG- inflammation, or infectious diseases. mediated TLR4 internalization and endocytic trafficking require additional investigation. Acknowledgments TLR4 translocation to the endosomes is required for TRIF- We thank Dr. Daniela A. Malide, National Heart, Lung, and Blood In- dependent IRF3 activation (11, 38). After transport to the early stitute Light Microscopy Core Facility, for skillful help with confocal endosome, TLR4 is sorted and targeted to a trans-Golgi network microscope imaging. pathway (43) and diverted to a lysosomal-degradation pathway (12, 25), which is required for resolution of the inflammatory Disclosures response. Minimal perturbation of any of these steps causes an The authors have no financial conflicts of interest. abnormal inflammatory response (12, 44). Indeed, recent studies showed that abnormal accumulation of TLR4 in early endosomes by inhibition of endocytosis or endosomal sorting leads to in- References creased LPS-induced activation of the NF-kB, MAPK, and IRF3 1. Akira, S., S. Uematsu, and O. Takeuchi. 2006. Pathogen recognition and innate pathways, which affect the downstream proinflammatory state (12, immunity. Cell 124: 783–801. 2. West, A. P., A. A. Koblansky, and S. Ghosh. 2006. Recognition and signaling by 45, 46). toll-like receptors. Annu. Rev. Cell Dev. Biol. 22: 409–437. Consistent with these reports, our results demonstrate that 3. Kawai, T., and S. Akira. 2010. The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors. Nat. Immunol. 11: 373–384. GMFG knockdown induced delayed TLR4 internalization and 4. Akira, S., and K. Takeda. 2004. Toll-like receptor signalling. Nat. Rev. Immunol. abnormal accumulation of TLR4 in early endosomes, which led to 4: 499–511. The Journal of Immunology 6103

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