TLRs induce autophagy in RAW 264.7 cells. A, activated TLR4 or TLR3 leads to autophagosome formation in RAW 264.7/GFP-LC3 cells. RAW 264.7 cells stably transfected with GFP-LC3 were treated with LPS (100 ng/ml) for 7 h to activate TLR4, and with poly(I·C) (20 μg/ml) for 7 h to activate TLR3. The cells were imaged by confocal microscopy. B, TLR ligands cause autophagy in RAW 264.7/GFP-LC3 cells. Pam₃CSK₄·3HCl (100 ng/ml) was used to stimulate TLR1. TLR3 was activated with poly(I·C) (20 μg/ml). TLR4 was engaged by LPS (100 ng/ml). TLR5 was activated with Flagellin (50 ng/ml). TLR6 was stimulated with MALP-2 (50 ng/ml). TLR7 was activated by ssRNA40 (10 μg/ml). TLR9 was activated by CpG oligodeoxy nucleotides (5 μg/ml). All ligands were incubated with the cells for 7 h. The percentage of GPF-LC3 positive cells with GFP-LC3 dots were counted by confocal microscope. The data shown represent mean ± S.D. from three independent experiments. C, activated TLR4 or TLR3 induces autophagy in RAW 264.7 cells based on the ratio of endogenous LC3-I to LC3-II as shown by immunobloting with anti-LC3 antibodies. LPS and poly(I·C) at the above concentrations were added into the cells for 16 h; and then the cellular lysates were immunoblotted with antibodies against LC3. D, representative electron micrographics of RAW 264.7 cells treated with LPS or poly(I·C) for 16 h. Arrows denotes autophagosomes.

Activated TLR4 recruits Beclin 1, MyD88, and Trif, and the death domain of MyD88 is required to associate with Beclin 1. A, an agonistic TLR4 antibody was used to stimulate RAW 267.4 cells and immunoprecipitate the TLR4-associated protein complex at the indicated time points. A matched control antibody was used for mock stimulation and immunoprecipitation. For the time 0 point non-stimulated RAW 267.4 cells were lysed, the TLR4 antibody was added and used to immunoprecipitate associated proteins. The washed immune complexes were analyzed by immunoblotting with the indicated antibodies. A portion of the cell lysates were saved and used to detect endogenous protein levels by immunoblotting with the indicated antibodies. B, MyD88 and the N terminus of MyD88 associated with Beclin 1. A FLAG-tagged MyD88, FLAG-tagged MyD88 death domain (NT), or FLAG-tagged MyD88 TIR domain (CT) was transfected into RAW 267.4 cells. An anti-FLAG monoclonal antibody was used for immunoprecipitation and immunoblotting. Anti-Beclin 1 antibodies were used for detecting the associated endogenous Beclin 1. C, Trif associates with Beclin 1. An anti-Au1 monoclonal antibody was used to immunoprecipitate expressed Au1-Trif. Endogenous Beclin 1 that co-immunoprecipitated was detected by anti-Beclin 1 antibodies. Similar results in two experiments performed.

Bcl2, MyD88, and Trif target Beclin 1 to regulate autophagy in RAW 264.7 macrophages. A, overexpression of Bcl-2 suppresses LPS (100 ng/ml, 7 h) and poly(I·C) (20 μg/ml, 7 h) leads to autophagy. RAW 267.4 cells were transfected with 0.2 μg of GFP-LC3 and 0.6 μg of Bcl2 plasmids for 24 h. The total amount of plasmid DNA was adjusted to 0.8 μg/transfection with the pcDNA3 vector. The percentages of GFP-LC3 positive cells with GFP-LC3 dots were counted. The results shown represent mean ± S.D. of three experiments. B, overexpression of MyD88 or Trif partially reverses the inhibition of Beclin 1-induced autophagy by Bcl2. RAW 264.7 cells were transfected with the following expression vectors: 0.2 μg of GFP-LC3, 0.3 μg of Beclin 1, 0.5 μg of Bcl-2, and MyD88, MyD88 death domain (NT), MyD88 TIR domain (CT), Trif, or Trif dominant negative. The total amount of plasmid DNA was adjusted at 1.5 μg/transfection with the pcDNA3 vector. The percentages of GFP-LC3 positive cells with GFP-LC3 dots were counted. The results shown are representative of one of three independent experiments performed.

Involvement of MyD88, Trif, and Beclin 1 in TLR3 and TLR4 triggered autophagy in RAW 264.7 macrophages. A, expressed MyD88, Trif, Beclin 1, or TRAM, but not SARM triggers autophagy. RAW 264.7 cells were transfected with 0.2 μg of GFP-LC3 and 0.6 μg of MyD88, Trif, Beclin 1, SARM, or TRAM plasmids for 24 h. The total amount of plasmid DNA was adjusted at 0.8 μg/transfection with pcDNA3 empty vector. The percentages of GFP-LC3 positive cells with GFP-LC3 dots were counted. The results shown represent mean ± S.D. from three independent experiments. B, a dominant negative form of MyD88 or Trif, and shRNAs targeted at MyD88, Trif, or Beclin 1 impair LPS-induced autophagy. RAW 264.7 cells were transfected with 0.8 μg of the indicated plasmids and 0.2 μg of GFP-LC3 as an autophagic marker for 24 h. The shRNA targeted at luciferase was used as a negative control. The cells were treated with LPS (100 ng/ml) for 7 h. The autophagic cells were calculated based on the percentage of GPF-LC3 positive cells with GFP-LC3 dots. The data shown are the mean ± S.D. from three independent experiments. C, dominant negative form of Trif or shRNAs targeted at Trif or Beclin 1 impairs poly(I·C)-induced autophagy. RAW 264.7 cells were transfected with 0.8 μg of the indicated plasmids and 0.2 μg of GFP-LC3 for 24 h. The shRNA that targets luciferase was used as a negative control. The cells were treated with 20 μg of poly(I·C) for 7 h. The autophagic cells were calculated as the percentage of GPF-LC3 positive cells with GFP-LC3 dots. The data shown are the mean ± S.D. from three independent experiments. D, reduced expression of MyD88, Trif, or Beclin 1 attenuates LPS-induced autophagy as determined by cleavage of endogenous LC3. RAW macrophages were transfected with 0.8 μg of the indicated shRNAs and again 24 h later. The luciferase shRNA was used as a negative control. The cells were treated with LPS (100 ng/ml) for 16 h. The levels of endogenous LC3-I and LC3-II were detected by immunobloting with anti-LC3 antibodies. Actin was used to verify equal sample loading. The data shown are one of two independent experiments performed. E, reduced expression of Trif or Beclin 1 impairs TLR3-induced autophagy as assessed by immunoblotting endogenous LC3. A double transfection of the shRNA constructs was performed. The data shown are one of two independent experiments. F, immune blots showing that the shRNAs targeting MyD88, Trif, or Beclin 1 mRNAs (two different sites) reduced expression of the corresponding protein.

MyD88 and Trif modulate the interaction between Bcl2 and Beclin 1. A, LPS or poly(I·C) stimulation facilitates the association of Beclin 1 with MyD88 and Trif. Lysates from RAW 264.7 cells treated with LPS (100 ng/ml) or poly(I·C) (20 μg/ml) for 4 h were immunoprecipitated (IP) with anti-Beclin 1 antibodies. The resulting immune complexes were analyzed with the indicated antibodies. B, TLR4 and TLR3 activated by LPS and poly(I·C), respectively, promote the association of MyD88 and Trif with Bcl2, and suppress the interaction between Bcl2 and Beclin 1. The cellular lysates from the above preparation and treatment were also immunoprecipitated with a Bcl-2 antibody. The resulting immune complexes and corresponding lysates were analyzed using the indicated antibodies. The induction of autophagy was monitored by immunoblotting with anti-LC3 antibodies. C, LPS stimulation promotes MyD88 association with Beclin 1. RAW 267.4 cells were exposed to LPS (100 ng/ml) for 4 h and the cellular lysates were immunoprecipitated with anti-MyD88 antibody. They along with their corresponding lysate were analyzed by immunoblotting using the indicated antibodies. Autophagy was monitored by LC3 immunoblotting. D, ssRNA40 stimulation promotes MyD88 association with Beclin 1 and decreases Bcl-2 binding to Beclin 1. RAW 267.4 cells were treated with ssRNA40 (10 μg/ml) for 4 h and the cellular lysates were immunoprecipitated with anti-Beclin 1, Bcl-2, or a MyD88 antibody. The indicated proteins were immunoblotted. E, overexpression of FLAG-MyD88 and FLAG-MyD88 death domain (NT), but not the FLAG-MyD88 TIR domain (CT) partially suppresses the interaction between Beclin 1 and Bcl-2. The indicated constructs were transfected into RAW 267.4 cells for 24 h, and then anti-Bcl-2 antibodies were used to immunoprecipitate Bcl2. Co-immunoprecipitated Beclin 1 was detected by immunoblotting. The indicated proteins in cell lysates were detected by immunoblotting. F, overexpression of Bcl-2 attenuates the association of Beclin 1 with MyD88 and Trif. Following transfection of the Bcl-2 expression vector cell lysates were subjected to immunoprecipitation with anti-Beclin 1 antibodies. The resulting immunoprecipitates and the corresponding cell lysates were analyzed by immunoblotting with the indicated antibodies. G, FLAG-tagged Beclin 1 L116A has a decreased association with Trif, MyD88, and Bcl2. Beclin 1 or Beclin 1 L116A was transfected into RAW 267.4 cells and 24 h later immunoprecipitations were performed with anti-FLAG antibodies. The immunoprecipitates were analyzed by immunoblotting with the indicted antibodies. The above results come from one of three independent experiments that gave similar results.