Lyn Delivers Bacteria to Lysosomes for Eradication through TLR2-Initiated Autophagy Related Phagocytosis

<div><p>Extracellular bacteria, such as <i>Pseudomonas aeruginosa</i> and <i>Klebsiella pneumoniae</i>, have been reported to induce autophagy; however, the role and machinery of infection-induced autophagy remain elusive. We show that the pleiotropic Src kinase Lyn mediates phagocytosis and autophagosome maturation in alveolar macrophages (AM), which facilitates eventual bacterial eradication. We report that Lyn is required for bacterial infection-induced recruitment of autophagic components to pathogen-containing phagosomes. When we blocked autophagy with 3-methyladenine (3-MA) or by depleting Lyn, we observed less phagocytosis and subsequent bacterial clearance by AM. Both morphological and biological evidence demonstrated that Lyn delivered bacteria to lysosomes through xenophagy. TLR2 initiated the phagocytic process and activated Lyn following infection. Cytoskeletal trafficking proteins, such as Rab5 and Rab7, critically facilitated early phagosome formation, autophagosome maturation, and eventual autophagy-mediated bacterial degradation. These findings reveal that Lyn, TLR2 and Rab modulate autophagy related phagocytosis and augment bactericidal activity, which may offer insight into novel therapeutic strategies to control lung infection.</p></div

Lyn plays an important role in recruitment of LC3 upon Pa infection.

<p>(A) MH-S cells were pretreated with CD (2.5 μg/ml, 30 min), and then infected with PAO1 (MOI = 10, 1 h). Cell viability was tested by MTT assay. (B) Cells treated as above were lysed for CFU assay. (C) Cells lysates were performed for immunoblotting of pLyn, Lyn and LC3. (D, E) MH-S cells were transfected with LC3-RFP plasmid for 24 h and pretreated with CD (2.5 μg/ml, 30 min). The cells were then infected with PAO1-GFP (MOI = 10, 1 h). Confocal microscopy images were used to detect LC3 puncta upon Pa infection. LC3 puncta in each cell were counted and the percentage of LC3⁺/Pa⁺ colocalization (LC3-RFP and PAO1-GFP) is shown. Data are derived from 100 cells in each sample. (F) MH-S were pretreated with CD (2.5 μg/ml, 30 min) or rapamycin (500 nM, 12 h) and cells were lysed for immunoblotting of pLyn, Lyn and LC3. (G) MH-S cells were transfected with Ctrl or Lyn siRNA for 24 h, and treated with rapamycin (500 nM, 12 h). Cells lysates were performed for immunoblotting of pLyn, Lyn and LC3. (H, I) MH-S cells were co-transfected with LC3-RFP and Ctrl or Lyn siRNA for 24 h. The cells were treated with rapamycin (500 nM, 12 h). Confocal microscopy images were used to show LC3 puncta. LC3 puncta in each cell were counted. The percentage of LC3⁺ events (cell with more than five LC3 puncta was considered as positive) is shown. Data are derived from 100 cells in each sample. (J) MH-S cells were treated with Zymosan (10 μg/ml, 1 h). Cell lysates were performed for immunoblotting of pLyn, Lyn and LC3. (K) MH-S cells were co-transfected with Lyn-GFP and LC3-RFP for 24 h. The cells were treated with Zymosan (10 μg/ml, 1 h). Confocal microscopy images were used to show Lyn or LC3 puncta. (L) LC3 puncta in each cell were counted, and the percentage of LC3⁺ events is shown as above. Data are derived from 100 cells in each sample. (M, N) MH-S cells were transfected with Ctrl, Rubicon or ULK1 siRNA for 24 h. The cells were infected with PAO1 (MOI = 10, 1 h). Cell viability and phagocytic abilities were tested by MTT or CFU assays as above. (O) Cell lysates from above were used for immunoblotting to detect pLyn, Lyn and LC3, respectively. (P) MH-S cells were co-transfected with Ctrl, Rubicon or ULK1 siRNA, respectively along with LC3-RFP for 24 h. The cells were infected with PAO1-GFP (MOI = 10, 1 h). LC3 puncta in each cell were counted, and the percentage of LC3⁺ events is shown as above. Data are derived from 100 cells in each sample. All data are representative as means+SD of three independent experiments. One-way ANOVA (Tukey’s post hoc); *, p<0.05; **, p<0.01. Scale bar = 5 μm.</p

Lyn regulate infection-induced autophagy through Rab5.

<p>(A) MH-S cells were transfected with Rab5-RFP for 24 h. Cells were infected with PAO1-GFP (MOI = 10, 1 h). Immunostaining was used to detect pLyn. Pa surrounded by Rab5 and pLyn was detected by confocal microscope. Scale bar = 5 μm. (B) The percentage of Rab5⁺/Pa⁺/pLyn⁺ colocalization (Rab5-RFP, PAO1-GFP, and pLyn puncta) is shown. Data are derived from 100 cells in each group. (C) MH-S cells were infected with PAO1 (MOI = 10, 1 h). Co-IP was performed to detect the protein interactions between Lyn and Rab5. (D) MH-S cells were pretreated with PP2 (5 nM, 30 min). Phagosomes were isolated and lysed for immunoblotting to detect pLyn and Rab5. (E, F) MH-S cells were co-transfected with Rab5-RFP and LC3-GFP as well as Lyn siRNA for 24 h, respectively. Cells were infected with PAO1 as above. CLSM imaging showed the localization (different puncta). Data are derived from 100 cells for each sample. Scale bar = 5 μm. (G, H) MH-S cells were transfected with Rab5-RFP or Rab5-DN-RFP plasmid for 24 h. Cells were infected with PAO1-GFP as above. Cell viability was determined using MTT assay and phagocytosis was performed by CFU assay. (I, J) CLSM imaging showed the localization of Rab5 and Pa. Scale bar = 5 μm. Rab5 puncta were counted in each cell. The percentage of Rab5⁺/Pa⁺ events (cell with colocalized puncta of Rab5-RFP and PAO1-GFP) is shown. Data are derived from 100 cells in each group. (K) MH-S cells were transfected with Ctrl or Lyn siRNA for 24 h, respectively. Whole cell lysates were immunoprecipitated (IP) with beads coated with Lyn antibody and immunoblotted with actin antibody. (L, M) Cells were infected with PAO1-GFP (MOI = 10) for different time. Polymerized actin was stained with rhodamine-phalloidin. Confocal microscopy showing rhodamine-phalloidin staining (red) around GFP-expressing Pa. Phagosomes containing degraded bacteria are heavily labeled for polymerized actin (white arrowheads). Scale bar = 20 μm. Internalized Pa and colocalized puncta were counted in each cell. Data are derived from 100 cells in each group. One-way ANOVA (Tukey’s post hoc); *, p<0.05.</p

Autophagy is important for Lyn-dependent elimination of Pa in macrophages.

<p>(A) MH-S cells were transfected with Ctrl, Atg5 or Beclin1 siRNA for 24 h, respectively. Immunoblotting was performed to verify the knock down of these two genes. (B) After Atg5 or Beclin1 was knocked down, MTT assay was used to determine the cell viability upon PAO1 infection (MOI = 10, 1 h). (C) CFU assay was performed to test the phagocytosis ability from above samples. (D) Cells above were collected and lysed for immunoblotting to measure the phosphorylation level of Lyn. (E) Quantification of pLyn and LC3-II level in D is shown. (F) MH-S cells were transfected with LC3-RFP for 24 h. Cells were infected with PAO1-GFP (MOI = 10, 1 h). Immunostaining was used to detect pLyn. Pa surrounded by LC3 and pLyn was detected by confocal microscope. Scale bar = 5 μm. (G) LC3 puncta in each cell were counted. The percentage of LC3⁺/Pa⁺/pLyn⁺ events (cell with colocalized puncta of LC3-RFP, PAO1-GFP, and pLyn) is shown. Data are derived from 100 cells in each sample. (H) MH-S cells were infected with PAO1 as above. After 1 h, cells were washed and subjected to polymyxin B for another 1 h. 10 h later, cell viability was determined by MTT assay. (I) Clearance assays were performed by counting CFU. (J) MH-S cells were infected with PAO1 for 1 h or 12 h as above. Cell lysates were performed for immunoblotting to test the invaded bacteria using Pa antibody. (K) Quantification of Pa protein in J is shown. All data are representative as means+SD of three independent experiments. One-way ANOVA (Tukey’s post hoc); *, p<0.05; **, p<0.01.</p

TLR2 is involved in infection-induced autophagy.

<p>(A) MH-S cells were infected by PAO1 (MOI = 10), HKPa (MOI = 10) and Pa LPS (100 ng/ml) for 1 h. HKPa was obtained by heating PAO1 at 60°C for 60 min. Cell lysates were collected and immunoblotting of pLyn, Lyn and LC3 were performed. (B, C) MH-S cells were transfected with LC3-RFP plasmid for 24 h. CLSM imaging showed infection by PAO1, HKPa and Pa LPS induced LC3 puncta. LC3 puncta in each cell were counted. Data are derived from 100 cells in each sample. (D) MH-S cells were transfected with Ctrl, TLR2 or TLR4 siRNA for 24 h, respectively. Immunoblotting were performed to prove the knock down of TLR2 or TLR4. (E, F) After TLR2 or TLR4 knock down as above, the cells were infected with PAO1 (MOI = 10, 1 h). MTT assays were used to measure the cell viability and CFU assays were used to measure phagocytosis. (G, H) MH-S cells were co-transfected with LC3-RFP and Ctrl, TLR2 or TLR4 siRNA for 24 h, respectively. Cells were then infected with PAO1 as above. CLSM imaging showed infection-induced LC3 puncta. LC3 puncta in each cell were counted. Data are derived from 100 cells in each sample. (I) MH-S cells were transfected with Ctrl or TLR2 siRNA for 24 h, respectively. Cells were infected with PAO1 as above. Cell lysates were performed for immunoblotting to detect pLyn, Lyn and LC3. (J) The HEK293 cells were stably transfected with a pUNO-TLR2 plasmid which expresses TLR2. TLR2 stable expression cells and 293/null cells were infected with PAO1 as above. Immunoblotting was performed to detect TLR2, pLyn, Lyn and LC3. (K, L) 293 cells were transiently transfected with LC3-RFP. After 24 hours cells were infected with PAO1-GFP (MOI = 10, 1 h). Arrows indicate significant LC3 puncta upon Pa infection using CLSM imaging. LC3 puncta in each cell were counted and the percentage of LC3⁺/Pa⁺ events (cell with colocalized puncta of LC3-RFP and PAO1-GFP) is shown. Data are derived from 100 cells in each sample. (M, N) MH-S cells were pretreated with Pam₃CSK₄ (300 ng/ml), and infected with PAO1 as above. Cells were homogenized for CFU and immunoblotting to detect TLR2, pLyn, Lyn and LC3. All data are representative as means+SD of three independent experiments. One-way ANOVA (Tukey’s post hoc); *, p<0.05; **, p<0.01. Scale bar = 5 μm.</p

Lyn kinase activities are required to facilitate autophagy related phagocytosis.

<p>(A) MH-S cells were transiently transfected with LC3-RFP. After 24 hours cells were infected with PAO1-GFP (MOI = 10, 1 h). Cells were fixed and stained with anti-pLyn antibody for immunofluorescence detection. Arrows indicate significant LC3 puncta colocalized with pLyn and Pa using CLSM imaging. Scale bar = 5 μm. (B) The statistic results from samples in A show LC3 puncta in each cell that are infected with Pa for different time. At 1 h post-infection, polymyxin B was added (1 h) to kill residual bacteria. The percentage of LC3⁺/Pa⁺/pLyn⁺ events (cell with colocalized puncta of LC3-RFP, PAO1-GFP, and pLyn) is shown. Data are derived from 100 cells in each sample. (C, D) MH-S cells were infected with PAO1 (MOI = 10, 1 h). After infection, cells were processed and examined by TEM. Arrow indicates autophagosome with double membranes containing internalized bacteria. The percentage of internalized Pa surrounded by double membrane is shown. Data are from 20 cells in each sample. Scale bar = 1 μm. (E) MH-S cells were infected with PAO1 as above. After infection, cell lysates were processed for co-IP to examine the interactions between Lyn and Atg12-Atg5, LC3 and Pa. (F) GST tagged Lyn peptide fragments were used to study <i>in vitro</i> association of Lyn with autophagy related proteins. (G) MH-S cells were infected with PAO1 (MOI = 10, 1 h) and then lysed for pull-down assay. GST-Lyn 1–230 containing both SH3 and SH2 domains shows association with Atg5-Atg12, LC3 and Pa. (H, I) MH-S cells were transfected with Lyn WT, DN (Lyn K275D), and constitutively active (Lyn Y598F) plasmid for 24 h and then treated with PAO1 as above. MTT assays were used to assess cell viability. CFU assays were used to measure phagocytosis. (J) Cells above were lysed for immunoblotting to detect the protein levels of pLyn, Lyn and LC3. (K, L) MH-S cells were transfected with Lyn WT, DN, and constitutively active plasmid for 24 h and then treated with PAO1-GFP (MOI = 10, 1 h). Confocal microscopy images were used to show significant LC3 puncta staining upon Pa infection. LC3 puncta in each cell were counted, and the percentage of LC3⁺/Pa⁺ events (cell with colocalized puncta of LC3-RFP and PAO1-GFP) is shown. Data are derived from 100 cells in each sample. Scale bar = 5 μm. All data are representative as means+SD of three independent experiments. One-way ANOVA (Tukey’s post hoc); *, p<0.05; **, p<0.01.</p