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TLR4 Endocytic Trafficking in Macrophages Modulates TLR4

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TLR4 Endocytic Trafficking in Macrophages Modulates TLR4 Glia Maturation Factor-γ Negatively Modulates TLR4 Signaling by Facilitating TLR4 Endocytic Trafficking in Macrophages 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 http://www.jimmunol.org/content/190/12/6093 Downloaded from References This article cites 47 articles, 19 of which you can access for free at: http://www.jimmunol.org/content/190/12/6093.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 28, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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 actin depolymerization factor/cofilin superfamily protein that is expressed in inflammatory cells, has been implicated in mediating neutrophil and T cell migration, but its function in macrophage 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 endocytosis 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 proteins 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
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