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

Extracellular bacteria, such as Pseudomonas aeruginosa and Klebsiella pneumoniae, 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.


Introduction
Gram-negative bacteria, such as Pseudomonas aeruginosa (hereafter Pa) and Klebsiella pneumoniae (Kp) are formidable threats to human health, imposing huge healthcare costs worldwide. Pa is the fourth most commonly isolated nosocomial pathogen, accounting for 10% of all hospital-acquired infections, while Kp is the third most commonly isolated pathogen from the blood of sepsis patients [1,2]. Increased incidence of antibiotic resistance and lack of effective treatment approaches further limit current interventional options. Alveolar macrophages (AM) are on the front line of host defense with a potential role in eliminating bacterial pathogens by phagocytosis and inflammatory responses [3,4]. Despite decades of extensive research efforts, the role of AM in phagocytosis and clearance of extracellular bacteria remains incompletely understood, which hinders the development of effective therapeutic strategies.
Autophagy is a highly conserved homeostatic mechanism for degrading bulk cellular components during starvation, or other scenarios, to provide the cell with essential nutrients. It has been linked to a wide variety of normal physiological processes including energy metabolism, organelle turnover, growth regulation, and aging [5]. Impaired autophagy can affect the process of various diseases, such as cardiomyopathy, cancer, and infection [6]. Innate immune effectors, such as toll like receptors (TLRs), are important for host defense against pathogens through initiation of phagocytosis and inflammatory response [7]. Autophagy may be modulated following the recognition of conserved pathogen-associated molecular patterns (PAMPs), which interact with host pattern recognition receptors (PRRs), such as TLRs [8,9]. Autophagy can be induced in murine macrophages by several TLR ligands, including poly (I:C) (TLR3), LPS (TLR4) and single strand RNA (TLR7) [7]. Interactions between phagocytes, including AM, and bacteria may critically influence the fate of both pathogens and phagocytes through multiple signaling cascades [10]. However, little is known about whether there is interaction between autophagy and phagocytosis during bacterial invasion.
Further characterization of the mechanistic underpinnings required to launch and execute immune defenses to eliminate bacterial infection is expected to significantly improve our knowledge of bacterial pathogenesis, thereby providing insight into the design of novel and effective therapeutics. One of the central themes in effective host defense is to understand how host cells counteract invasive bacteria, especially participating in the transport of bacteria to lysosomal killing environments for proteolytic digestion. A recent study of the intracellular bacterium Mycobacterium showed that the autophagy adaptor SQSTM1 (p62) can enhance delivery of bacterial cytosolic components and increase bacterial killing following phagocytosis [11]. Autophagy adaptors, such as SQSTM1, NDP52 and optineurin, were shown to mediate LC3 recruitment to the ubiquitinated substrate during ubiquitin-dependent xenophagy. Formation of the isolation membrane takes place in the proximity of the early phagosomes. Subsequently, the autophagosome engulfs the pathogen-containing phagosome. In contrast to the double-membraned autophagosome, which is not formed in LC3-associated phagocytosis (LAP), the phagosomal membrane is impacted directly by LC3 [12,13].
Prior studies implicated that the Src kinase Lyn initiates FcγR-mediated phagocytosis and participates in the process of post-phagosome formation by interacting with cytoskeletal proteins [14,15]. In the case of the extracellular bacterium Pa, we discovered that Lyn, lipid rafts, and TLR2 may play a role in phagocytosis [16,17]. Here, we demonstrate that TLR-2 is required for inducing Lyn activity in host defense against Pa infection by facilitating autophagosome maturation. We hypothesized that Lyn-mediated phagocytosis may link autophagy to phagocytosis in a TLR2-Lyn dependent manner. We report that Lyn is a critical upstream signaling component, which expands the concept of general xenophagy [12,18]. In addition, we dissected the molecular and cellular bases regarding how Lyn and autophagy contribute to innate immunity through the eventual degradation of bacterial components.

Lyn deficiency decreases phagocytosis and autophagy against Pa infection
To analyze the expression pattern of autophagy-related genes, we determined their mRNAs in mouse alveolar macrophage MH-S cells after Pa infection using an autophagy based RT 2 Profiler PCR Arrays (catalogue number: PAMM-084Z, Qiagen, Valencia, CA). The array analysis revealed that many autophagy related mRNAs (i.e., LC3-II, Atg4C, and Atg16L2) were upregulated in macrophages (S1A and S1B Fig, S1 Table), suggesting that autophagy may be involved in bacterial infection. To dissect whether the critical E1 enzyme Atg7 was required for host defense against Pa, we targeted Atg7 by siRNA in MH-S cells or isolated primary AM from wild type (WT) and atg7 knockout (atg7 -/-) mice (S2A Fig). Phagocytosis or clearance assays were performed after shorter (1 h) or longer (12 h) periods of Pa infection [19]. A prior study suggested that Lyn may be a critical factor during Pa infection [17]. MH-S cells and mouse primary AM were used to assess phagocytosis and bacterial clearance using CFU assays. After Lyn knockdown, the cell viability was determined using a MTT assay, which showed no difference upon Pa infection (Fig 1A), while bactericidal activity was impaired by Lyn deficiency (Fig 1B). Next, we transfected MH-S cells with Lyn-GFP, scrambled siRNA or Lyn siRNA, and infected the cells with another fluorescent-emitting and lipid-affinitive strain, Pa-Cherry. We observed significant Lyn aggregates (puncta) around the invading Pa by confocal laser scanning microscopy (CLSM) (Fig 1C). CLSM imaging demonstrated that internalization by macrophages was decreased with Lyn deficiency at the early time, suggesting the involvement of Lyn in phagocytosis ( Fig 1D). After removing the floating and surface bacteria by polymyxin B treatment at 1 h, the internalized Pa was killed with time. Lyn deficient AM showed decreased bactericidal ability (Fig 1D). Endogenous LC3 conversion into phosphatidylethanolamine-conjugated LC3-II was drastically inhibited by Lyn siRNA in MH-S cells as determined by immunoblotting (Fig 1E and 1F). Further, Lyn knockdown impeded LC3 puncta formation (Fig 1G and 1H). To reflect the physiological relevance, we also isolated primary AM to determine whether Lyn regulates autophagy in these cells. Similarly, significantlydecreased LC3 conversion was found in primary AM from Lyn -/mice upon Pa infection ( Fig  1I and 1J); and primary AM from Lyn -/mice also exhibited a marked reduction in LC3 puncta as detected by CLSM imaging (Fig 1K and 1L). MLE-12 cells, a murine lung epithelial cell line that does not appreciably phagocytose or clear bacteria, also showed LC3 puncta upon Pa infection, which was also impaired by Lyn deficiency or by perturbing autophagy (3-MA addition) (S2F Fig). In vivo experiments were then used to determine autophagy induction upon Pa infection. Lyn -/mice exhibited increased mortality as compared to WT mice after Pa infection ( Fig 1M). The increased bacterial burdens in the lungs also implied severely impaired bacterial killing in Lyn -/mice (S2G Fig). Lung histological analysis revealed that induction of LC3 puncta was decreased (Fig 1N), and pathophysiological tissue damage was more severe in Lyn deficiency following Pa infection (S2H Fig). Since PMN play an important role in innate immunity to Pa infection [20], PMN migration into infected lungs was measured and found to be increased in Lyn -/mice as determined by immunostaining with Ly6G (S2I Fig). These  Fig 1E is shown. (G) The LC3-RFP plasmid was transfected into MH-S cells along with Ctrl or Lyn siRNA for 24 h. Cells were then infected with PAO1-GFP (MOI = 10, 1 h). Confocal microscopy images were taken to determine LC3 puncta staining upon infection. Scale bar = 5 μm. (H) LC3 puncta in each cell were counted and the percentage of LC3 + /Pa + events (cell with at least one LC3 puncta was considered to be positive) is shown. Data are derived from 100 cells in each sample. (I) Primary AM from WT or Lyn -/mice were collected. Cells were infected with PAO1 (MOI = 10, 1 h), and cell lysates were collected and performed for immunoblotting of Lyn and LC3. (J) Quantification of LC3-II expression in Fig 1I is shown. (K) Primary AM were transfected with LC3-RFP for 24 h and infected with PAO1-GFP (MOI = 10, 1 h). Confocal microscopy images show significant LC3 puncta upon Pa infection. Scale bar = 5 μm. (L) Quantifications of LC3 puncta in each cell and the percentage of LC3 + /Pa + events are shown as above. (M) WT mice and Lyn -/mice were infected with 1×10 7 CFU of PAO1 (6 mice/group). Survival test was represented by Kaplan-Meier survival curves (p = 0.0065; 95% confidence interval, 0.8-4.5; Log-rank Test). (N) Lungs from above were removed and paraffin sectioned for immunostaining of LC3. Scale bar = 50 μm. The data represent means+SD from three experiments. One-way ANOVA (Tukey's post hoc); *, p<0.05. results indicate that Lyn is involved in an autophagy related phagocytosis to benefit Pa clearance in macrophages, which may preserve host cell viability after infection.

Lyn is required for autophagy induction upon Pa infection
To distinguish whether Lyn plays a role in Pa infection-induced autophagy, we first blocked internalization of Pa into macrophages using cytochalasin D (CD), a phagocytosis inhibitor. While CD had no effect on cell viability upon Pa infection (Fig 2A), its addition inhibited phagocytosis by macrophages ( Fig 2B). Interestingly, CD decreased LC3 conversion and phosphorylation (Tyr297) of Lyn (pLyn) upon Pa infection (Figs 2C and S3A). CLSM imaging also indicated that CD decreased LC3 puncta upon Pa infection (Fig 2D and 2E). To define the relationship of CD with autophagy, rapamycin was used as a positive control to induce autophagy. CD did not induce LC3 conversion or phosphorylation of Lyn. Moreover, CD did not inhibit rapamycin-induced LC3 conversion (Figs 2F and S3B). Next, siRNA interference was used to further determine the role of Lyn in autophagy. As shown in Figs 2G and S3C, Lyn deficiency did not affect rapamycin-induced autophagy without infection. CLSM imaging also confirmed that Lyn deficiency did not affect rapamycin-induced LC3 puncta (Fig 2H and 2I). These data suggest that Lyn is required for Pa infection-induced autophagy. We hypothesized that Lynmediated phagocytosis is associated with autophagy upon Pa infection. Zymosan (yeast wall extract) was used to treat macrophages, which did not alter cell viability (S3D Fig). LC3 conversion was found to be induced by Zymosan treatment. However, Zymosan did not induce Lyn phosphorylation ( Fig 2J and S3E Fig). In addition, CLSM imaging showed LC3 puncta formation while no Lyn puncta were formed upon Zymosan treatment of macrophages (Fig 2K and  2L). Furthermore, Gram-positive bacterium S. pyogenes (Sp) infection was used to determine the induction of LC3 puncta and Lyn puncta by CLSM imaging (S3F Fig). Collectively, these data indicated the role of Lyn in bacterial infection-induced autophagy. To distinguish conventional autophagy from LAP, the upstream preinitiation complex (ULK1/2, which is required for autophagy but not LAP), and Rubicon (which mediates LAP but not autophagy) were knocked down by siRNA interference strategy, respectively (S3G Fig) [21]. Knockdown of Rubicon does not affect the cell viability upon Pa infection ( Fig 2M); however, ULK1 interference resulted in decreased bacterial phagocytosis by macrophages ( Fig 2N). In addition, ULK1 interference led to decreased pLyn level and also LC3 lipidation as compared to Ctrl siRNA transfected cells (Figs 2O and S3H). CLSM imaging quantitation also showed decreased LC3 puncta by ULK1 deficiency upon Pa infection ( Fig 2P). However, Rubicon interference did not affect Lyn phosphorylation while slightly decreased LC3 lipidation upon Pa infection, with only limited phagocytosis reduction, indicating that LAP is less relevant to this infection model (Fig 2M-2P, S3H Fig). To further determine that LC3-lipidation and dot formation occur upon Pa infection, chloroquine was used to inhibit lysosome activities. Lyn phosphorylation was found with or without chloroquine. Furthermore, we observed that LC3-II conversion had accumulated with chloroquine treatment upon Pa infection determined by immunoblotting (S3I and S3J Fig).

Lyn kinase activities are needed to facilitate infection-induced autophagy
Interestingly, CLSM imaging showed that LC3 recruitment and phosphorylation of Lyn localized together with invading Pa (Fig 3A and 3B). To unravel whether the phagocytosis of Pa is involved in autophagosome formation, we detected newly formed autophagosomes using transmission electron microscopy (TEM) on Pa-infected MH-S cells. Pa-containing autophagosomes with double membranes were identified by TEM (Fig 3C and 3D), providing additional evidence that Pa-containing autophagosomes had formed, which is also termed as xenophagy. Next, co-immunoprecipitation (co-IP) was performed to determine the interaction of Lyn with autophagy-related proteins. As shown in Fig 3E, we observed that Lyn could bind well to Atg12-Atg5 and LC3 complex upon Pa infection. In addition, we found that Lyn associated with Pa ( Fig 3E), which suggested that Lyn may play a role in regulating autophagosome formation upon bacterial infection. To further understand the molecular mechanism in Lyn-mediated autophagosome formation, we asked which domain(s) of Lyn is required for 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.
doi:10.1371/journal.ppat.1005363.g002 interaction with the functionally or structurally relevant proteins. Lyn-GST peptides with distinct functional domains (see schematic, Fig 3F) were prepared as described previously [16,22]. The Lyn-GST fragments were coated on immobilized glutathione agarose beads to pull-down interacting partners from MH-S cell lysates and probed for Atg12-Atg5, LC3, or Pa, respectively. The presence of both Src homology 2 (SH2) and SH3 domains of Lyn were found to be required for interaction with signaling proteins within autophagosomes, whereas the kinase domain was dispensable for the interaction upon Pa infection (Fig 3G, S4A and S4B Fig).
These data further suggested that Lyn may interact with critical autophagic proteins localized in autophagosomes through SH2 and SH3 binding regions.
We next investigated whether this interaction specifically occurred in macrophages with strong phagocytosis. Primary AM and epithelial cells (MLE-12) were infected with Pa and cell lysates were applied for pull-down by Lyn-GST fragments. Although SH2 and SH3 domains of Lyn were required for interactions with autophagic proteins and binding to Pa in macrophages, no apparent Lyn-Pa interaction in MLE-12 cells was observed (S4C and S4D Fig), which may be due to different mechanisms of uptaking Pa by epithelial cells. To confirm that Lyn activation was responsible for the underlying process, MH-S cells were transfected with Lyn WT, Lyn K275D (dominant negative), and Lyn Y508F (constitutively active) constructs and treated with Pa. The transfection of different plasmids had no effect on the cell viability upon infection (Fig 3H), whereas Lyn K275D transfected cells showed impaired phagocytic ability upon bacterial infection (Fig 3I). Lyn activation was detected by immunoblotting for pLyn. While Lyn Y508F plasmid-transfected cells showed enhanced activity of Lyn kinase, Lyn K275D-transfected cells showed decreased activity as compared to Lyn WT-transfected cells (Figs 3J and S4E). LC3-II conversion was found to be inhibited in Lyn K275D transfected cells upon Pa infection (Figs 3J and S4E). Furthermore, LC3 puncta revealed that Lyn kinase activities were well correlated with Pa-induced autophagy in macrophages (Fig 3K and 3L). Our data also showed that PP2 (a commonly used Lyn inhibitor) reduced infection-induced autophagy (S4F and S4G Fig). Overall, the results demonstrated that the role of Lyn in infection-induced autophagy was dependent on its kinase activities.

TLR2 is involved in Lyn-initiated autophagy upon Pa infection
We next attempted to determine whether a single bacterial component (i.e. LPS), can induce autophagy. Interestingly, heat-killed Pa (HKPa) and LPS all caused LC3-II conversion to a similar extent as Pa, as detected by immunoblotting (Figs 4A and S5A). On the other hand, CLSM fluorescence microscopy showed that HKPa or LPS also induced LC3 puncta (Fig 4B and 4C). Importantly, we also observed increased phosphorylation of Lyn by LPS pretreatment (Figs 4A and S5A). These results indicate that Pa or LPS is potentially sufficient to induce autophagy in macrophages. Based on our previous observations [17], we probed the role of TLR2 and TLR4 as upstream signals to initiate autophagy. After TLR2 or TLR4 knockdown (Fig 4D), MH-S cells were infected with Pa. Both TLR2 and TLR4 deficiency impaired the phagocytosis of Pa ( Fig  4E), while cell viability was not altered (Fig 4F). However, only TLR2 knockdown showed impaired LC3 puncta formation upon bacterial infection (Fig 4G and 4H). Immunoblotting also showed that TLR2 deficiency inhibited LC3 conversion and Lyn phosphorylation upon bacterial infection (Figs 4I and S5B). These results suggest that TLR2 may play a role in autophagy induction during Pa infection (although other TLRs' role cannot be excluded at this time). To delve into the involvement of TLR2, HEK293 cells with stable TLR2 expression were infected by Pa. Although these cells are not phagocytes, Lyn activation (shown as pLyn level) or autophagy induction (shown as LC3 conversion or LC3 puncta) was much stronger in cells overexpressing TLR2 than vector control cells (Fig 4J-4L (Figs 4N and S5F). These results suggested that Lyn may serve as a bridge between TLR2 and autophagy after Pa infection.

Autophagy plays a role in Lyn mediated bacterial clearance
To assess whether autophagy has a role on Lyn-dependent elimination of Pa in macrophages, we knocked down Atg5 and Beclin1 using siRNA (Fig 5A). Atg5 or Beclin1 has little effect on 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 Pam 3 CSK 4 (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. cell viability upon Pa infection ( Fig 5B); however, phagocytosis was inhibited by down-regulating these two genes ( Fig 5C). Immunoblotting also showed that the phosphorylation of Lyn was decreased by Atg7, Atg5 or Beclin1 deficiency (Fig 5D and 5E). CLSM imaging also showed the LC3 puncta and pLyn level was dampened by a deficiency in either of these autophagic genes (Fig 5F and 5G). The clearance of phagocytized bacteria was also dampened by inhibiting Atg5 or Beclin1 in AMs upon Pa infection (Fig 5H and 5I). We then performed immunoblotting to verify the digestion of the invading bacteria and found that when autophagic genes were knocked down, the degradation of the bacteria proteins was blocked over time (Fig 5J and 5K). All these results suggest that autophagy plays an important role in Lyn-mediated phagocytosis and clearance upon bacterial infection.

Lyn regulates infection-induced autophagy through Rab and cofilin
To further assess the phagocytosis process, we elucidated whether Rab5 (early endosome marker) is involved in Lyn-mediated phagocytosis and subsequent bacterial clearance. We transfected MH-S cells with Rab5-RFP plasmid and infected the cells with Pa for different times. Immunostaining was next performed to probe pLyn to evaluate the internalized bacteria using CLSM microscopy. The number of phagocytized Pa was increased at the early time of infection. Interestingly, Rab5 and Lyn were both found to be surrounded with invading Pa, which implied that Rab5 may play a role in conjunction with Lyn in phagocytosis (Fig 6A and  6B). Next, co-IP was performed to confirm the interaction of Lyn and Rab5 (Figs 6C and S6A). To this end, we isolated phagosomes after Pa infection and the results showed that pLyn was found in the same fraction as Rab5 (e.g., phagosomes), while PP2 pretreatment impaired this process (Fig 6D). To test our hypothesis that Lyn is involved in Rab5-mediated autophagy related phagocytosis of Pa, we co-transfected Rab5-RPF, LC3-GFP with Ctrl or Lyn siRNA into MH-S cells. After Lyn knockdown, the formation of autophagosomes was impaired upon Pa infection (Fig 6E and 6F). Similar results were found with Rab7 (late endosome marker). We observed an interaction of Lyn with Rab7 (S6A Fig; Lyn deficiency also decreased autophagosome-lysosome formation upon Pa infection (S6B and S6C Fig). These data suggest that Lynmediated phagocytosis may be dependent on the conserved endocytosis pathway involving Rab families. We then used dominant negative plasmids to down-regulate Rab5 and Rab7 and found that Rab5-DN-RFP transfection did not affect cell viability (Fig 6G), while dampened the phagocytosis (Fig 6H, S6D and S6E Fig) and colocalization of Rab5 with Pa (Fig 6I and 6J). Importantly, Rab7-DN-RFP transfection also significantly inhibited phagocytosis and colocalization of Rab7 and Pa inside the autolysosome (S6F and S6G Fig). Collectively, these findings suggest that Lyn, together with Rab family members, participated in the transportation of invading bacteria in phagocytosis and clearance upon infection.
To determine whether Lyn is involved in actin remodeling in macrophages, we performed co-IP and found that Lyn interacted with actin after Pa infection (Fig 6K). Lyn siRNA-transfected cells showed significantly decreased polymerized F-actin indicated by phalloidin staining (around Pa containing phagosomes) vs. Ctrl siRNA-transfected cells at both 2 h and 8 h (Fig  6L and 6M). Also, flotillin-1 expression, which is required for nucleation of actin on phagosomal membrane, correlated with the internalization of Pa, and was inhibited by Lyn siRNA interference (S6H and S6I Fig)

Lyn mediates fusion of lysosome with Pa-containing autophagosome
To determine the critical role of Lyn in delivering bacterial components and in enabling intracellular traffic, a process required for autophagosome-lysosome fusion, LAMP1-RFP was transfected into macrophages to track lysosomes. We found that both LAMP1 and pLyn were colocalized with internalized Pa at 8 h post infection (Fig 7A and 7B). To biochemically characterize the role of Lyn and autophagy in Pa trafficking inside the cell, we isolated phagosomes from Pa-infected cells using a sucrose gradient method [16]. Horse radish peroxidase (HRP) was used to determine phagosome distribution and Pa Ab was used to show phagosome. We found that both Atg12-Atg5 and LC3 were colocalized with Lyn, and were shifted into early phagosome, late phagosome or phagolysosome fractions following infection (Fig 7C and 7D). Importantly, Pa bacterial proteins were detected using polyclonal Pa antibody in phagolysosome (45%; fractions 3, 4, 5), early phagosome (30%; fraction 6) and late phagosome (25%; fractions 7, 8,9), while negligible in other fractions as determined by immunoblotting (Fig 7C). The presence of LAMP1 in fractions 9 and 10 indicates the autophagosome fusion with lysosome and a potential role of autophagy in bacterial delivery and clearance (Fig 7C). CLSM imaging showed that the colocalization of LC-3 and Lyn was drastically impaired by Lyn knockdown (Fig 7E and 7F). Importantly, proteolytic assay also showed that the degradation of the phagocytized bacteria (Figs 7G-7I, S7A), while the lysosome inhibitor chloroquine prevented the digestion of the invading bacteria (Figs 7J and S7B). Finally, we used another clinically significant Gram-negative bacterium, K. pneumoniae (Kp), to elucidate whether the autophagy related phagocytosis is a general phenomenon during bacterial infection. As expected, Kp infection also induced LC3 puncta around internalized bacteria, which was inhibited by Lyn deficiency or 3-MA (S7C-S7G Fig). To further dissect that autophagy-dependent innate immunity is generally important for immunity to P. aeruginosa or K. pneumoniae infections, additional strains (PAK, PA14, Kp, and Kp-Xen 39) were used to determine whether manipulation of autophagy impacts bacterial survival. Reduced bacterial killing was caused by autophagy inhibition or Lyn deficiency, indicating that Lyn-assisted autophagy is a general pathway for innate immunity against these microorganisms. These results collectively demonstrate that Lyn has an essential role in Gram-negative bacteria-induced autophagy, facilitating bacterial clearance. Fig 7K illustrates a model delineating the role of Lyn in Pa-induced autophagy related phagocytosis.

Discussion
The current study demonstrates that TLR2/Lyn signaling plays a critical role in recruitment of autophagic components to bacteria-containing phagosome. We show that Lyn mediates the formation of the membrane isolation in the proximity of the Pa-containing phagosome in AM. Our data further reveal that Lyn transmits signaling for cytoskeleton protein-mediated intracellular transport following TLR2 activation, resulting in a merge between autophagosome and phagosome. This autophagosome-lysosome fusion helps eliminate the internalized Pa, leading to enhanced bacterial clearance. Further, blocking autophagy and/or depleting Lyn hamper the delivery of bacteria into lysosomes, thereby dampening bacterial eradication. Taken together, we characterize TLR2 and Lyn as new regulators of bacteria-induced autophagy and phagocytosis both in vitro and in vivo, providing new insight into the molecular mechanism of immune responses against acute bacterial infection.
Autophagy induced by intracellular bacterial infections has garnered increasing interest and has been shown to play crucial roles in host defense in immunological cells [23,24]. This autophagic role is relevant to the bacterial fates after phagocytes engulf some critical pathogenic bacteria, such as M. tuberculosis, L. monocytogenes, S. enterica and S. flexneri [24][25][26][27]. Previous reports have defined that macrophages with Atg7 deficiency could express enhanced scavenger receptor to phagocytize and retain M. tuberculosis [28]. However, whether autophagy can influence the uptake of extracellular bacteria, such as Pa or Kp, is largely unknown. A recent report showed that Pa infection enhanced autophagy [29], and that Atg7 -/mice showed enhanced susceptibility to infection and impaired bacterial clearance [29]. However, there is no in vivo evidence to show reduced bacterial clearance associated with autophagy deficiency in Lyn -/mice [30,31]. Using mouse models, we show that autophagy related proteins contribute to xenophagy of extracellular bacteria Pa or Kp, thus affecting phagocytosis of host macrophages. Our data implicate that the role of autophagy varies with bacteria.
Previous studies indicate that PRRs may be activated in a PAMP-specific manner. The TLR5-mediated response was shown to be induced by flagellin, while LPS appears to link to the TLR4-mediated response [32,33]. LPS is one of the important structural components in the outer membrane of Gram-negative bacteria. CD14 facilitates the transport of LPS to the TLR4/ MD-2 complex and regulates LPS recognition [34]. However, TLR2 and TLR4 recognize different bacterial cell wall components [35,36]. We show that Pa induces autophagy and that HKPa initiates host inflammatory responses through TLR2. We noted that LPS, a TLR4 ligand, also induced autophagy. Previous studies indicated that TLR2-mediated recruitment of LC3 to phagosomes may promote phagolysosome fusion [18]. Consistent with this observation, we showed that Lyn boosts phagocytosis of macrophages and facilitates subsequent intracellular trafficking processes.
Our previous studies showed that Lyn plays a role in Pa infection [16,17,31]. However, whether Lyn can regulate autophagy has remained elusive. Others show that blocking autophagy sensitizes prostate cancer cells towards Src family kinase inhibitors [37]. Interestingly, Lyn is reported to increase the survival of glioblastoma cells under starvation-induced autophagy [38]. Here, we identify a role of Lyn in initiating autophagy during Pa infection. Unlike LAP, typical double-membraned autophagosomes that contain bacteria are formed in our study, which is also specifically relevant to the activity of Lyn.
We also noted that blocking autophagy by 3-MA impaired phagocytosis. mTOR is a major negative regulatory axis of autophagy, thus inhibition of mTOR and its associated pathways can induce autophagy. We also found that rapamycin increases autophagy to elevate innate immune defense against Pa infection. These findings indicate that Lyn facilitates phagocytosis through autophagic mechanisms and serves as a link between phagosomes and autophagosomes. Thus, we delineate a novel mechanism by which Lyn mediates autophagy-related phagocytosis to benefit host against Pa.
We also demonstrate that Lyn's activity is required for inhibiting Pa infection, as depletion of Lyn and/or Atg7 in macrophages resulted in increased Pa growth. Hence, the participation of Lyn and Atg7 suppressed Pa replication, whereas the absence of Lyn or Atg7 or other autophagic proteins (Atg5 and Beclin1) allowed for an uncontrolled bacterial growth. Murine macrophages lacking Lyn are defective in migration and in phagocytosis [17]. These observations suggest that the uptake of Pa may be impaired in Lyn-deficient macrophages. Previous studies suggest that intracellular growth of bacteria requires the halt of phagolysosome fusion [39]. This intracellular trafficking defect is observed in permissive macrophages, and most bacteria-containing vacuoles fuse with lysosomes to clear bacteria [40]. How autophagosome-lysosome fusion is modulated upon Pa infection is poorly understood. Our findings demonstrate that Lyn may modulate the delivery of bacteria to lysosomes, facilitating autophagosome maturation and subsequent bacterial clearance.
Phagosomes bind microtubules and actin filaments and migrate to interact with other compartments within the cell [41]. Our studies indicate that Lyn is involved in polymerization of Factin, a cofilin-dependent process. Rab5, an early endosome marker, enables the recruitment of early endosomes to form nascent phagosomes and is also required for the recruitment of Rab7 (a late endosome marker), thus promoting phagolysosome fusion [42]. By isolating phagosomes to biochemically identify which phagosomal constituents interact with Lyn, we found that both Rab5 and Rab7 interact with Lyn, which may be downstream regulators following Lyn-mediated autophagy related phagocytosis. Thus Rab5 and Rab7 may be required for delivering bacterial components to lysosomes in Lyn-mediated Pa phagocytosis and autophagosome-lysosome fusion.
In summary, our studies represent autophagic benefit in host defense against Pa through a TLR2/Lyn axis. The contribution of Lyn to autophagy is found to facilitate the removal of intracellular bacteria within macrophages. Our results also demonstrate that TLR2 activates Lyn. Identification of both Lyn and TLR2 as novel regulatory factors in autophagy may implicate their therapeutic potential for control of Gram-negative bacterial infection.

Ethics statement
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocols were approved by the Institutional Animal Care and Use Committee at the University of North Dakota, School of Medicine (Assurance Number: A3917-01). Dissections and injections were performed under anesthesia that was induced and maintained with ketamine hydrochloride and xylazine, and all efforts were made to minimize suffering.
Mice atg7-deficient (floxed atg7 -/-) mice were provided by Dr. Youwen He at Duke University and these mice were originally constructed by Masaaki Komatsu at Tokyo Metropolitan Institute of Medical Science [43]. To generate mice with a monocyte-specific knockout of Atg7, we have generated myeloid-specific conditional knockout (KO) mice by cross-breeding atg7 flox/flox with lyz 2tm1(Cre)lfo>/j mice (Jackson Laboratory, Bar Harbor, ME) [44]. The knockout was induced by injecting 0.1 mg of tamoxifen (Sigma, St Louis, MO) daily for 5 days [45]. lyn KO mice were provided by Dr. S. Li at University of Massachusetts. Age and sex matched wild-type (WT) mice (C57BL/6J) were used as controls [31].

Cells
MH-S (ATCC CRL-2019) were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (HyClone Laboratories, Logan, UT) and 100 U/ml of penicillin/streptomycin (P/ S, Life Technologies, Rockville, MD) antibiotics in a 37°C incubator with 5% CO 2 . Mouse AM were isolated by bronchoalveolar lavage (BAL) [46]. In brief, trachea was cannulated with a 20-gauge catheter; 0.9 ml BAL buffer was instilled, flushed four times, and retrieved. A total of 3.0 ml BALF was retrieved from each mouse and cytospin slides prepared with 0.5 ml BALF were stained by HEMA-3 (Fisher, Rockford, IL) to enumerate leukocyte subtypes based on their cellular and nuclear morphological properties. After centrifugation at 2, 000 rpm, AM cells were resuspended and cultured in RPMI 1640 medium as above. MLE-12 (ATCC CRL-211) were cultured in HITES medium as above. Stable TLR2 expression HEK293 cells (ATCC CRL-157) using a pUNO-TLR2 plasmid were obtained from InvivoGen (San Diego, CA) and cultured in DMEM medium as above.

Infection experiments
Bacteria were grown overnight in lysogeny broth (LB) at 37°C with vigorous shaking. Next day, the bacteria were pelleted by centrifugation at 8, 000×g and resuspended in 10 ml of fresh LB broth, in which they were allowed to grow until the mid-logarithmic phase. Thereafter, the optical density (OD) at 600 nm was measured, and the density was adjusted to 0.25 OD (1 OD = 1×10 9 cells/ml). Before infection, cells were washed once with PBS, and replaced with antibiotic-free medium immediately [47]. Cells were infected by Pa at multiplicity of infection (MOI) of 10:1 (bacteria-cells ratio) for 1 h. After infection, macrophages were washed 3 times with PBS, extracellular bacteria were removed by addition of 100 μg/ml polymyxin B and left in incubation for another 1 h. Phagocytosis was evaluated by counting colony forming unit (CFU) [16]. Bacterial clearance by AM cells was also determined using the CFU assay after 1 h infection, following with 1 h polymyxin B incubation and then 10 h culture in antibiotics free medium to gauge eradication of the internalized bacteria, indicating clearance of bacteria [48]. For in vivo experiments, 40 mg/kg ketamine was used for anesthesia. Mice were intranasal instilled with 1×10 7 CFU of Pa (suspended in 25 μl PBS). Mice were sacrificed 24 h post infection. The lungs were collected for paraffin section. For survival experiments, mice (6/group) were infected as above and monitored till moribund.

RT-PCR array
Real-time PCR profiling of mRNAs were conducted on a SYBR Green-based, RT 2 Profiler PCR Array System (Cat# PAMM-084Z, Qiagen, Valencia, CA). The array includes 84 key genes that encode components of the molecular machinery and key regulators modulating autophagy in response to both extracellular and intracellular signals. Expression of mRNAs was detected by QuantiTect SYBR Green RT-PCR Kit (Qiagen, Valencia, CA). The separate well 2-ΔΔCt cycle threshold method was used to determine relative quantitative levels of individual mRNA, and these were expressed as the fold difference to GAPDH, respectively.

Immunoblotting analysis
Rabbit polyclonal antibodies against, Atg7, Atg12-Atg5, TLR2, Lyn, pcofilin-1, cofilin-1, flotillin-1, actin and mouse polyclonal antibodies against LC3, GAPDH were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit monoclonal antibody against phosphor-Lyn (Tyr297) was obtained from Cell Signaling Technology (Danvers, MA). Rabbit monoclonal antibody against flagellin type b and Pa were kindly provided by Dr. Gerald Pier of Harvard Medical School. The samples from cells were lysed and quantified. The lysates were boiled for 10 min, and added with protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN). The supernatants were collected and 20 μg of each sample were loaded onto 12% SDS-polyacrylamide gel electrophoresis (PAGE) and electrophoresed for protein resolution. The proteins were then transferred to polyvinylidine difluoride membranes (Thermofisher, Rockford, IL) and blocked for 1 h at room temperature using 5% non-fat milk blocking buffer. Membranes were incubated for 2 h at room temperature with appropriate first antibodies diluted at 1:1000 in 5% bovine serum albumin (BSA) immunoblotting antibody buffer. After washing (three times, 10 min once) with washing solution, the membranes were incubated for 60 min at room-temperature with horseradish peroxidase-conjugated secondary antibody (Rockland Immunochemicals, Gilbertsville, PA) diluted 1:3000. Signals were visualized using an enhanced chemiluminescence detection kit (SuperSignal West Pico, Pierce) [46]. Co-immunoprecipitation (co-IP) is performed using Protein A/G-argarose beads and then mix with the related antibody. The target proteins were detected using immunoblotting as above.

Confocal microscopy and indirect immunofluorescence staining
Cells were grown either on coverslips in a 24-well plate or in glass-bottomed dishes (MatTek, Ashland, MA). Rabbit monoclonal antibody against Ly6G was bought from Abcam (Cambridge, MA). For immunostaining, the cells or slide tissues were fixed in 3.7% paraformaldehyde, permeabilized with 0.2% Triton X-100 in PBS and incubated with blocking buffer containing 2% BSA for 30 min. Samples were incubated with primary antibodies at 1/500 dilution in blocking buffer for 1 h and washed three times. After incubation with appropriate fluorophore-conjugated secondary antibodies, the cover slips were mounted on slides with Vectashield mounting medium. The images were captured using an LSM 510 Meta confocal laser scanning microscope (CLSM, Carl Zeiss Micro Imaging, Dublin, CA), and processed using the software provided by the manufacturer [50].

GST-Lyn pull-down assay
GST-Lyn constructs with different functional domains were originally obtained from Dr. O. Miura (Tokyo Medical and Dental University, Tokyo, Japan) [22] and transformed into BL21-DE3 strain of E. coli. The GST-Lyn fragments were extracted using immobilized glutathione columns following the manufacturer's instructions [16]. Equal whole cell lysates were incubated with GST-Lyn peptide for protein interactions. The pull-down products were analyzed by immunoblotting with specific Abs.

Phagosome isolation
Cell lysates were processed for phagosome isolation, as previously described [51]. Briefly, the cell homogenates were separated by sucrose gradient density centrifugation ranging from 90 to 5% of sucrose cushion. The phagosomes were heavy and separated at 65% density, whereas lysosomes were found at the interface of 35 and 5% densities. Fractions (1.1 ml) were collected from top (total 10 fractions) corresponding to the density gradient.

Transmission electron microscopy (TEM)
TEM was employed for identifying autophagosomes using modified Karnovsky's fixative [52]. Images were taken and processed according to our previous reports [30].

Statistical analysis
All experiments were performed in triplicate and repeated at least three times. Data are presented as changes compared with the controls from the three independent experiments. Results are shown as means±SD. One-way ANOVA (Tukey's post hoc) was used for statistical analysis. Differences were accepted as significant at p<0.05. (TIF) S1 Table. Micro-array data of autophagy related genes expression. (DOCX)