Tim-4 internalizes apoptotic cells through Axl-mediated signal transduction

Tim-4 internalizes apoptotic cells through Axl-mediated signal transduction | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Tim-4 internalizes apoptotic cells through Axl-mediated signal transduction Daeho Park, Byeongjin Moon, Jaeseon Jeon, Eunhye Oh, Jinwook Han, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7580891/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract A pivotal step in phagocytosis of apoptotic cells, called efferocytosis, is phagocytic recognition of phosphatidylserine (PS) on dying cells, primarily via PS receptors. Among them, Tim-4 has been studied to elucidate signaling since its identification. However, how apoptotic cells secured by Tim-4 are internalized remains still unclear. Here, we reveal that Axl-mediated signal transduction is necessary for Tim-4 to ingest apoptotic cells. We found correlated expression patterns for Tim-4 and Axl across various tissues, and biochemical and cell biological analyses showed that Tim-4 interacted most strongly with Axl among the TAM receptor family. Mechanistically, the immunoglobulin variable (IgV) domain of Tim-4 interacted with two distinct domains within the extracellular region of Axl. Co-expression of Tim-4 and Axl synergistically promoted Tim-4-mediated efferocytosis. Crucially, disrupting the interaction or pharmacologically or genetically blocking Axl signaling abolished the synergistic effect of Axl on Tim-4-mediated efferocytosis. These findings collectively demonstrate that during Tim-4-mediated efferocytosis, Axl acts as a key signaling relay, biochemically interacting with Tim-4 to transmit apoptotic cell recognition signals into phagocytes, thereby enabling efficient apoptotic cell ingestion. Biological sciences/Cell biology/Cell death/Apoptosis Biological sciences/Cell biology/Cell signalling Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Efferocytosis, phagocytosis of apoptotic cells, is an essential process for both development and homeostasis [ 1 , 2 , 3 ]. A hallmark of this process is the ability of phagocytes to recognize apoptotic cells, distinguishing them from viable cells. The ability is mediated by specific molecules expressed on apoptotic cells and phagocytes. Apoptotic cells present unique molecular markers on their surface that differentiate them from live cells, while phagocytes express corresponding receptors. One of the best-known ligands on apoptotic cells is phosphatidylserine (PS), which is normally confined to the inner leaflet of the plasma membrane in live cells but becomes exposed on the outer surface during apoptosis [ 4 , 5 , 6 , 7 , 8 , 9 ]. The exposed PS is recognized by phagocytes either directly or indirectly. Receptors capable of direct PS recognition, known as PS receptors, include BAI1, Tim-4, Tim-1, and Stabilin-2 [ 10 , 11 , 12 , 13 , 14 , 15 ]. In contrast, TAM receptor tyrosine kinases (Tyro3, Axl, and Mertk) and integrins bind to PS indirectly through bridging molecules such as Gas6 and Mfge8 [ 16 , 17 , 18 , 19 ]. Among PS receptors, Tim-4, also known as T-cell immunoglobulin and mucin domain-containing protein 4 (Timd4), has attracted significant attention in the field of efferocytosis due to its specific expression in macrophages and the notable impairment in efferocytosis observed upon its loss [ 20 , 21 , 22 ]. Originally, Tim-4 was identified as a Tim-1-interacting protein with a role in T-cell proliferation [ 23 ]. Later, a screening of antibodies against mouse peritoneal macrophages revealed that Tim-4 functions as a PS-binding protein [ 11 ]. Subsequent studies on Tim-4 as a PS receptor elucidated the structure of the Tim-4–PS complex and highlighted its pivotal roles in efferocytosis and autoimmune diseases [ 12 , 24 , 25 ]. However, the downstream signaling pathways of Tim-4 remain largely unexplored. Some studies have suggested potential downstream signaling mechanisms, but it is generally accepted that Tim-4 itself is unable to directly transduce signals into phagocytes [ 26 ]. Given that Tim-4 lacks direct signaling, it relies on a co-receptor, biochemically interacting with Tim-4, to transduce signals into phagocytes. Alternatively, Tim-4 functions solely as a tethering receptor, securing apoptotic cells to facilitate their engulfment by another receptor. These two mechanisms are not mutually exclusive and may coexist. Mertk, a member of TAM receptor family, exemplifies the former mechanism, whereas integrins represent the latter [ 27 , 28 , 29 , 30 ]. The TAM receptor family is a unique group of receptor tyrosine kinases involved in various cellular processes such as cell migration, survival, and efferocytosis [ 17 , 31 ]. In the context of efferocytosis, TAM receptors bind to PS on apoptotic cells via bridging molecules such as Gas6 and Protein S, which induce dimerization of TAM receptors and facilitate the connection between apoptotic cells and TAM receptors. Both Gas6 and Protein S share a similar multidomain structure, where their SHBG domain interacts with the Ig like domains of TAM receptors and their Gla domain binds to PS. Gas6 can bind to all TAM family members, whereas Protein S preferentially binds to Mertk and Tyro3, with minimal affinity for Axl. Unlike Tim-4, TAM receptors can independently transmit signals for phagocytosis of the apoptotic cells they recognize [ 16 , 17 , 32 ]. Interestingly, our previous research demonstrated that Mertk biochemically interacts with Tim-4 through an interaction between the FnIII domain of Mertk and the IgV domain of Tim-4, thereby promoting Tim-4-mediated efferocytosis [ 33 ]. In this study, we investigated whether the other TAM receptors also biochemically interact with Tim-4 and, if any, play a cooperative role for Tim-4-mediated efferocytosis. Our results demonstrate that all TAM receptors interact with Tim-4, with Axl exhibiting the strongest interaction. The IgV domain of Tim-4 is responsible for its interaction with Axl, while Axl interacts with Tim-4 via both its FnIII and Ig like domains. Functionally, although Axl expression alone failed to promote efferocytosis, its co-expression with Tim-4 synergistically enhanced efferocytosis under serum-free conditions. Additionally, disrupting Tim-4–Axl interaction or blocking Axl signaling abolished the synergistic effect of Axl on Tim-4-mediated efferocytosis. Taken together, our findings suggest that Axl functions as a co-receptor, transducing signals generated by Tim-4 into phagocytes to induce apoptotic cell internalization through its biochemical interaction with Tim-4. Materials and Methods Cell culture and transfection HEK 293T cells were cultured and maintained in DMEM (Dulbecco’s Modified Eagle’s Medium) containing 10% FBS (Fetal Bovine Serum) and 1% PSQ (Penicillin-Streptomycin-Glutamine). LR73 CHO cells were cultured in α-MEM (Alpha’s Modified Eagle’s Medium) supplemented with 10% FBS and 1% PSQ. 293T and LR73 cells were transfected using CaPO 4 (following the lab’s protocol) and Lipofectamine 2000 (Invitrogen, Waltham, MA, USA) respectively. To obtain primary splenic macrophages, the spleens of C57BL/6 mice were collected and minced with a razor blade into 1mm 3 pieces and transferred to a culture dish. Thereafter, the medium was changed every two days to remove stromal cells and red blood cells. After one week, the adherent cells were detached using trypsin-EDTA and used for experiments. Mice C57BL/6 mice were purchased from Taconic Biosciences (Germantown, NY, USA). Axl −/− mice were gifted from Prof. Hyung-Sik Kang at Chonnam National University (Republic of Korea). 3 ~ 4-week-old mice were used to prepare apoptotic thymocytes. 8 ~ 10-week-old mice were used to obtain spleen macrophage. All mice were housed in a well-maintained animal facility under controlled temperature and humidity conditions. All mouse-related procedures were reviewed and approved by the Institutional Animal Care and Use Committee of the Gwangju Institute of Science and Technology, following the guidelines outlined by the National Institutes of Health for the ethical treatment and use of laboratory animals. Plasmids and reagents All plasmids in this study were constructed and sequenced to confirm their identity. Tim-4 ECR , Tim-4 IgV and Tim-4 Mucin plasmids used in this study have been previously described [ 33 ]. Axl ECR , extracellular region of Axl (residues 19–445), Axl Ig , immunoglobulin domain (residues 19–217), and Axl FnIII , fibronectin type III domain (residues 218–448) were cloned into the pEBG-GST vector or pEBB-GFP vector using PCR-based methods. The inhibitors and other reagents used in this study were R428 (S2841; Selleckchem, Huston, TX, USA), LY294002 (70920; Cayman Chemical Company), TAMRA-SE (C1171; Invitrogen, Carlsbad, MA, USA) pHrodo Red-SE (P36600; Invitrogen) Dexamethasone (D1756; Sigma-Aldrich, St. Louis, MO, USA). The antibodies used in the study were anti-Axl (AF854; R&D systems, Minneapolis, MN, USA), anti-Tyro3 (AF759; R&D systems), anti-Mertk (AF591, R&D Systems), anti-Tim-4 PE conjugated (12-5866-80; Invitrogen), anti-mouse F4/80 FITC clone:BM8 (123108, Biolegend, CA, USA) anti-FLAG (F1804, Sigma Aldrich, MO, USA), anti-HA (SC-7392, Santa Cruz biotechnology, Dallas, TX, USA), anti-HA (#3724, Cell signaling technology, Danvers, MA, USA), anti-GFP (ab290, Abcam, Cambridge, MA, USA), anti-GST (SC-138, Santa Cruz biotechnology) and normal goat IgG control (AB-108-C, R&D Systems). Fluorochrome-conjugated donkey anti-goat (Alexa Fluor 488, A-11055) and goat anti-rabbit secondary antibodies (Alexa Fluor 594, A-11037, Alexa Fluor 405, A-31556) were purchased from Invitrogen. Binding assay LR73 cells were plated on poly-D-lysine coated 18mm cover glass in a 12-well nontreated culture plate. One day after plating, the cells were transfected. The following day, cells were preincubated in serum-free alpha-MEM for 2 h and then incubated with 5-(and-6)-Carboxytetramethylrhodamine (TAMRA)-labelled apoptotic thymocytes suspended in serum free alpha-MEM at 4 o C for 2 h. After incubation, cells were washed with PBS, fixed and stained with PE-conjugated anti-Tim-4 and anti-Axl antibodies. Then cells were washed with PBS twice for 5 min each and stained with an Alexa fluor 488-conjugated secondary antibody for 1 h. Images were acquired by confocal microscopy (FV3000; Olympus, Tokyo, Japan). Spleen macrophages were plated on confocal dish. The day after seeding, cells were incubated with TAMRA-labeled apoptotic thymocytes in the same manner as previously described for use in the binding assay [ 34 ]. Then cells were washed with PBS, fixed and stained with a FITC-conjugated anti-F4/80 antibody at 4 o C for 1 day. After staining, cells were washed with PBS twice and mounted. Images were acquired by confocal microscopy (FV3000). Efferocytosis assay Efferocytosis assays were performed as previously described [ 35 ]. Briefly, LR73 cells were plated in a 24-well culture plate and transfected with the plasmids. One day after transfection, the cells were pre-incubated in serum-free alpha-MEM for 2 h and incubated with TAMRA-stained apoptotic thymocytes in serum-free alpha MEM in the presence or absence of purified proteins and inhibitors (LY294002, R428) for 2 h. The cells were washed with ice-cold PBS, trypsinized, analyzed using flow cytometry (BD FACS Canto II). Data were analyzed by FLOWJO software (FlowJo LLC, OR, USA). Spleen macrophages derived from indicated mice were pre-incubated in serum-free DMEM for 2 h and incubated with pHrodo-labeled apoptotic thymocytes in serum-free DMEM for 1 h. The macrophages were washed with ice-cold PBS, trypsinized and analyzed using flow cytometry. Immunofluorescence microscopy LR73 cells were plated on poly-D-lysine-coated 18mm cover glasses in a 12-well nontreated culture plate and transfected with indicated plasmids. The day after transfection, cells were fixed with 4% paraformaldehyde in PBS and blocked with 3% bovine serum albumin. Then, cells were immunostained with anti-Axl, anti-Tyro3, PE conjugated anti-Tim-4 antibodies at 4 o C for 1 day. After washing with PBS, cells were stained with an Alexa Fluor 488-conjugated anti-goat secondary antibody. Then cells were washed with PBS and mounted on slide glass using Duolink mounting media with DAPI. Images were obtained using a confocal microscope (FV3000). Immunoprecipitation and Immunoblotting 293T cells were plated on 100 mm cell culture dish and transfected with indicated plasmids using CaPO 4 . Then cells were lysed using lysis buffer (10 µg/mL AEBSF, 10 µg/mL aprotinin, 10 µg/mL pepstatin, 10 µg/mL leupeptin, 150 mM NaCl, 10 mM NaF, 10 mM NaPP, 1 mM Na3VO4, 1% Triton X-100, and 50 mM Tris (pH 7.6)). The lysates were incubated with indicated antibodies with Protein A/G-conjugated beads (10001D, 10003D, Thermo Fisher Scientific), FLAG-conjugated beads (A2220, Sigma Aldrich), or glutathione agarose beads (17-0756-01, GE healthcare, IL, USA). Bound proteins were loaded and separated on SDS-PAGE, transferred onto nitrocellulose membrane, and detected with appropriate antibodies. Bioinformatics The TAM receptors and Tim-4 expression profile in mouse tissues were retrieved from the UniProt database and visualized as a heatmap using GraphPad Prism 9. The expression of phosphatidylserine receptors in the mouse spleen was retrieved from the Expression Atlas database (EMBL-EBI). Metadata files, GSE41637 [ 36 ], GSE74747 [ 37 ], ERP000591 [ 38 ], and DRP001032 [ 39 ], were used to generate the expression heatmap of PS receptors. Co-localization index The Image of Axl or Tyro3 and Tim-4 expressing cells were obtained using a confocal microscope. The co-localization index was assessed by measuring the overlap between red fluorescence (Tim-4) and green fluorescence (Axl or Tyro3) using ImageJ 1.4. A color threshold was applied to define yellow pixels representing co-localized signals, as described in a previous study [ 33 ], and the co-localization index was calculated as the proportion of yellow pixels relative to the total number of pixels within the cellular area. Statistical analysis All data are shown as the mean value ± standard deviation (SD). Each experiment was performed independently at least three times. Statistical significance was analyzed by the Student’s two-tailed t-test for experiments involving two groups. A one-way analysis of variance (One-Way ANOVA) was performed for experiments involving three or more samples using GraphPad Prism 9 software (Prism 9, Graphpad Software, La Jolla, CA, USA). The significance was accepted when p < 0.05. Results The members of the TAM receptor family interact with Tim-4 Tim-4 is known to promote efferocytosis by cooperating with various proteins [ 27 , 29 , 33 , 40 ]. In a previous study, we reported that Tim-4 facilitates efferocytosis through biochemical interactions with proteins containing a FnIII domain [ 29 , 33 ]. Their FnIII domains bind to the IgV domain of Tim-4, mediating interaction with Tim-4 and leading to enhanced Tim-4-mediated efferocytosis. Interestingly, TAM receptors share high structural similarity, and all contain Ig like and FnIII domains and a kinase domain in their extracellular and intracellular region, respectively (Fig. 1 A). Besides, all TAM receptors are known to be receptors for efferocytosis as well. Nevertheless, it is still unclear whether Tyro3 and Axl also cooperate with Tim-4 for efferocytosis. To this end, we first investigated whether they biochemically associate with Tim-4. We overexpressed TAM receptors along with HA-Tim-4 in 293T cells and performed an immunoprecipitation assay. Hemagglutinin (HA)-tagged Tim-4 co-immunoprecipitated with all TAM receptor family members when immunoprecipitated using an anti-FLAG antibody (Fig. 1 B and Supplementary Fig. 1A). When a reverse immunoprecipitation assay was performed using an anti-HA antibody, both Axl or Tyro3 were still co-immunoprecipitated with HA-Tim-4 (Fig. 1 C, D, and Supplementary Fig. 1B, C). Notably, Axl exhibited the strongest binding to Tim-4 while Mertk showed relative weak interaction with Tim-4 among the receptors. Next, to evaluate their interaction within cells, FLAG-tagged Axl or Tyro3 was co-expressed with HA-tagged Tim-4 in phagocytes, and their subcellular localization was analyzed using confocal microscopy. Axl and Tyro3, along with Tim-4, were found to be exclusively expressed along the cell boundary, indicating their localization in the plasma membrane. Noticeably, the fluorescence signal from Axl markedly overlapped with that from Tim-4, to a greater extent than from Tyro3 (Fig. 1 E and F). The data suggest that all TAM receptors biochemically interact with Tim-4 with varying affinities, especially implying a more potent cooperative role of Axl for Tim-4 in efferocytosis. The IgV domain of Tim-4 mediates interaction with Axl. In addition to the strong interaction between Axl and Tim-4, a robust correlation was observed between Tim-4 and Axl expression across multiple tissues (Fig. 2 A). Given the greater relevance of Axl to Tim-4 compared to the other TAM receptors, we focused on Axl in subsequent studies concerning its relationship to Tim-4 in efferocytosis. Next, we investigated which regions of Axl and Tim-4 mediate their biochemical interaction. To this end, we generated various truncated mutants of Tim-4 and Axl to test whether the cytoplasmic tail or transmembrane domain of Tim-4 is required for the interaction (Fig. 2 B). Axl maintained its interaction with Tim-4-GPI, a glycosylphosphatidylinositol (GPI)-anchored Tim-4 mutant that lacks the cytoplasmic tail and transmembrane domain (Fig. 2 C and Supplementary Fig. 2A), indicating that the interaction between Axl and Tim-4 is mediated through their extracellular regions (ECRs). We then performed an immunoprecipitation assay to verify that the ECRs of both proteins are sufficient to mediate the interaction and confirmed interaction between the ECR of Axl and the ECR of Tim-4 (Fig. 2 D and Supplementary Fig. 2B). We further examined which domain(s) of the ECRs of both proteins mediate the interaction. Tim-4 contains one IgV domain and one mucin domain in its extracellular region, while Axl possesses two Ig like domains and two FnIII domains in its extracellular region (Fig. 1 A). Immunoprecipitation assays showed that the IgV domain of Tim-4 co-immunoprecipitated with the ECR of Axl, whereas the mucin domain did not (Fig. 2 E and Supplementary Fig. 2C), indicating that the IgV domain of Tim-4 is necessary for the interaction. Unexpectedly, however, both the Ig like and FnIII domains of Axl co-immunoprecipitated with the Tim-4 ECR (Fig. 2 F and Supplementary Fig. 2D), which contrasts with the previously reported interaction between Tim-4 and Mertk, where only the FnIII domain of Mertk binds to Tim-4. These results indicate multiple binding sites on Axl for Tim-4, which may lead to a stronger interaction of Axl with Tim-4 than that of Mertk or Tyro3. Tim-4 with Axl promotes efferocytosis synergistically. Next, we investigated the functional significance of the biochemical interaction between Axl and Tim-4 in efferocytosis. First, to determine whether Axl plays a role during Tim-4-mediated efferocytosis, we examined whether their biochemical interaction changes during efferocytosis. We co-incubated apoptotic cells with phagocytes overexpressing Tim-4 under serum-free conditions and assessed interaction of Tim-4 with endogenous Axl. Interestingly, without apoptotic cells, no detectable interaction between Tim-4 and endogenous Axl was observed. However, upon apoptotic cell stimulation, interaction between Tim-4 and endogenous Axl was induced (Fig. 3 A and Supplementary Fig. 3A), implying that Axl is involved in Tim-4-mediated efferocytosis. To further confirm this, we overexpressed Tim-4 and Axl in phagocytes and performed an efferocytosis assay. We performed experiments under serum-free conditions without bridging molecules (e.g., Gas6) to ensure that Axl does not contribute to efferocytosis by itself, and serum-free conditions were used in all subsequent experiments. Despite the low basal efferocytosis level under serum-free conditions, phagocytes expressing Tim-4 exhibited significantly enhanced efferocytosis whereas the cells expressing Axl alone failed to promote efferocytosis compared to the control cells. Especially, phagocytes co-expressing Tim-4 and Axl showed far more efficient efferocytosis than those expressing Tim-4 alone, as measured by the percentage and MFI (mean fluorescence intensity, an indicator of the relative number of apoptotic cells per cell) of phagocytes engulfing apoptotic cells (Fig. 3 B, C and Supplementary Fig. 4A). This efficient efferocytosis by phagocytes expressing both Tim-4 and Axl did not result from their altered ability to recognize apoptotic cells because the binding of apoptotic cells to phagocytes was comparable in cells expressing Tim-4 alone and those co-expressing Tim-4 and Axl (Fig. 3 D), implying Axl facilitates the internalization of apoptotic cell during Tim-4-mediated efferocytosis. Collectively, these data suggest that Axl transduces signals to promote the internalization of apoptotic cells secured by Tim-4 through the interaction with Tim-4 during efferocytosis. Blocking Axl signaling impairs Tim-4-mediated efferocytosis If Axl is indeed a signal transducer for the internalization of apoptotic cells during Tim-4-mediated efferocytosis, then blocking the interaction or Axl signaling should impair Tim-4-mediated efferocytosis. In our previous study, we have shown that the FnIII domain of Mertk binds to the IgV domain of Tim-4 and its soluble form (Mertk FnIII ) disrupts the interaction of Tim-4 with Mertk. Similarly, Mertk FnIII also abrogated the interaction between Axl and Tim-4 (Fig. 4 A and Supplementary Fig. 5A). When phagocytes were treated with Mertk FnIII , efferocytosis was impaired to a comparable extent in both Tim-4-expressing phagocytes and Tim-4/Axl co-expressing phagocytes. However, efferocytosis remained unaffected in control cells or Axl-alone expressing cells (Fig. 4 B), indicating that interaction of Axl with Tim-4 is necessary for the cooperative role of Axl for Tim-4-mediated efferocytosis. Next, to assess the effect of Axl signaling on Tim-4-mediated efferocytosis, we blocked Axl signaling using two different approaches. First, we generated a truncated mutant of Axl lacking its cytoplasmic tail (Axl DCT ), unable to mediate intracellular signal transduction, and tested its effect on Tim-4-mediated efferocytosis. Efferocytosis by phagocytes co-expressing Axl DCT and Tim-4 was not enhanced and comparable to that by phagocytes expressing Tim-4 alone (Fig. 4 C). As an alternative second approach to impair Axl signaling, we used R428 or LY294002, which specifically inhibits Axl or an Axl downstream molecule, PI3K, respectively. When phagocytes were treated with the inhibitors, efferocytosis by phagocytes expressing Tim-4 alone or Tim-4 and Axl was notably inhibited to a degree that was indistinguishable between the two groups (Fig. 4 D and E). These data indicate that Axl serves as a receptor that relays signals into phagocytes to promote the internalization of apoptotic cells secured by Tim-4 during efferocytosis. Tim-4-mediated efferocytosis by splenic macrophages requires Axl. The spleen is an organ where old or damaged red blood cells from the circulation are removed by splenic macrophages. We confirmed the high expression of Tim-4 and the dominant expression of Axl among TAM receptors in the spleen (Fig. 2 A). In addition, of all the PS receptors, Tim-4 was found to be predominantly expressed in the spleen, supported by publicly available gene expression databases (Fig. 5 A). Specifically, splenic macrophages showed high Axl expression, relatively low Mertk expression, and undetectable Tyro3 expression (Fig. 5 B). Therefore, to further validate the synergistic role of Axl for Tim-4-mediated efferocytosis in a more physiologically relevant system, we used splenic macrophages. We first evaluated the ability of wild-type ( WT ) and Axl-deficient ( Axl -/- ) splenic macrophages to recognize apoptotic cells. WT and Axl -/- splenic macrophages did not show a difference in their ability to bind to apoptotic cells (Fig. 5 C). However, efferocytosis by Axl -/- splenic macrophages was significantly less efficient than WT splenic macrophages (Fig. 5 D). Interestingly, this superior efferocytosis by WT splenic macrophages to Axl -/- splenic macrophages was abolished when treated with an Axl specific inhibitor, resulting in indistinguishable efferocytosis between WT and Axl -/- splenic macrophages (Fig. 5 E). These results imply that Axl transduces signals for the internalization of apoptotic cells secured by Tim-4 on splenic macrophages. In summary, our findings demonstrate that Axl serves as a signal transducer for Tim-4 in efferocytosis, promoting the internalization of apoptotic cells secured by Tim-4 through its interaction with Tim-4. Discussion When Tim-4 was first reported as a phosphatidylserine (PS) receptor recognizing PS during efferocytosis, its downstream signaling remained unclear. Subsequent studies demonstrated that even a truncated mutant of Tim-4 lacking a cytoplasmic tail could promote efferocytosis, leading to the classification of Tim-4 as a tethering receptor that cannot directly transmit signals [ 26 , 27 ]. Several studies have since proposed a two-step engulfment process, in which Tim-4 facilitates efferocytosis by coordinating with other engulfment receptors such as integrins [ 40 ]. Additionally, a co-receptor capable of biochemically interacting with Tim-4 to mediate signal transduction has been identified, significantly advancing our understanding of Tim-4 downstream signaling [ 29 , 33 ]. In this study, we investigated the biochemical interactions and functional roles of other TAM receptor family members that are structurally and functionally similar to Mertk, a previously reported binding partner of Tim-4. As expected, Axl and Tyro3, which share a high structural similarity with Mertk, both interacted with Tim-4. We found that the IgV domain of Tim-4 and the FnIII domain of Axl mediate the interaction. While the Mertk-Tim-4 interaction involves only the FnIII domain of Mertk binding to Tim-4, in the case of Axl, both its Ig like and FnIII domains bind to the extracellular region of Tim-4. This may contribute to Axl forming the strongest interaction with Tim-4 among TAM receptors. Additionally, given that the Ig-like domain of Axl is structurally and sequentially similar to the IgV domain of Tim-4, the Ig like domain of Axl can interact with its own FnIII domain, providing insights into the inhibition-activation mechanism of Axl. Further research on this interaction could be valuable in the future. All TAM receptor family members have been implicated in efferocytosis [ 41 , 42 , 43 ]. However, their expression patterns differ: while Mertk and Axl exhibit similar expression patterns to Tim-4, Tyro3 shows distinct differences. Specifically, in many resident macrophages, Tim-4, Axl, and Mertk are expressed, whereas Tyro3 is not. Despite confirming the biochemical interaction between Tim-4 and Tyro3 in this study, the physiological role of Tyro3 in Tim-4-mediated cellular events remains unclear and warrants further investigation. During TAM receptor-mediated efferocytosis, TAM receptors recognize apoptotic cells via Gas6 or ProS, leading to the activation of the PI3K-AKT signaling pathway [ 44 , 45 , 46 ]. This activation subsequently induces Rac activation and cytoskeletal rearrangement, allowing apoptotic cells to be internalized [ 17 ]. Therefore, in Tim-4-mediated efferocytosis, apoptotic cells recognized by Tim-4 appear to be internalized through the activation of the PI3K-AKT signaling pathway via a biochemical interaction with Axl. In this study, we found that apoptotic cell stimulation under serum-free conditions induces the biochemical interaction between Tim-4 and Axl, suggesting that this interaction may contribute to Axl activation. The interaction between the FnIII domains and Ig like domains may act as a self-inhibitory mechanism for Axl. During Tim-4-mediated efferocytosis, this interaction-mediated self-inhibition of Axl could be relieved and activated upon Tim-4 binding to the domains of Axl. Investigating how the interaction between the Ig like domains and FnIII domains of Axl affects its activation could be an intriguing topic for future research. In conclusion, this study demonstrates that Axl synergistically promotes Tim-4-mediated efferocytosis by transducing apoptotic cell recognition signals into phagocytes through biochemical interaction with Tim-4, effectively substituting for the inability of Tim-4 to transduce signals into phagocytes. Given that both Tim-4 and Axl are associated with various immune disorders, our findings provide valuable insights for the development of therapeutic strategies targeting related diseases. Declarations Conflict of Interest The authors declare no competing interests. Author contributions Conceptualization, B.M. and D.P.; Methodology, B.M., J.J., E.O., J.H., J.L., E.H., C.L., and D.J.; Formal analysis, B.M., J.J., E.O., J.H., J.L., and D.P.; Resources, B.M. and J.J.; Writing, B.M. and D.P.; Funding acquisition, B.M. and D.P. Acknowledgements This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Ministry of Science and ICT and Ministry of Health & Welfare, Republic of Korea (No. RS-2025-00558157, RS-2024-00466906, and RS-2024-00510912) and by GIST research Institute (GRI) IIBR. The confocal imaging was performed using instruments provided by the GIST Advanced Institute of Instrumental Analysis (GAIA). We sincerely thank Prof. Hyung-Sik Kang at Chonnam National University for the generous gift of Axl −/− mice. Availability of Data and Materials Further information and requests for materials should be directed to and will be fulfilled by the lead contact, Daeho Park ( [email protected] ). Plasmids generated in this study will be distributed upon request to other investigator under a Material Transfer Agreement. References Arandjelovic S, Ravichandran KS. Phagocytosis of apoptotic cells in homeostasis. Nat Immunol 2015, 16(9): 907–917. 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08:55:39","extension":"html","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":133111,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7580891/v1/f354b9756dc220dfa49082d3.html"},{"id":93665539,"identity":"22b8c492-a0a7-48a4-8610-4046068642cf","added_by":"auto","created_at":"2025-10-16 08:55:38","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":594221,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTAM receptors interact with Tim-4.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e Schematic diagram of the TAM receptor family and Tim-4. Ig, immunoglobulin; FnIII, fibronectin type III; PKD, protein tyrosine kinase domain; IgV, Immunoglobulin variable. \u003cstrong\u003eB\u003c/strong\u003e-\u003cstrong\u003eD\u003c/strong\u003e 293T cells were transfected with the indicated plasmids. 2 d after transfection, the cells were lysed, and FLAG (\u003cstrong\u003eB\u003c/strong\u003e) or HA (\u003cstrong\u003eC\u003c/strong\u003e, \u003cstrong\u003eD\u003c/strong\u003e) tagged proteins were precipitated with anti-FLAG (\u003cstrong\u003eB\u003c/strong\u003e) or HA (\u003cstrong\u003eC\u003c/strong\u003e, \u003cstrong\u003eD\u003c/strong\u003e) antibody-conjugated agarose beads, respectively. Bound proteins were detected with the indicated antibodies. IP, immunoprecipitation; TCL, total cell lysates. The blots are representative of at least three independent experiments. \u003cstrong\u003eE\u003c/strong\u003e LR73 cells transfected with HA-Tim-4 and Axl-FLAG or Tyro3-FLAG were fixed, stained with DAPI, anti-HA, and anti-Axl or anti-Tyro3 antibodies, and observed by confocal microscopy. Arrows indicate where two proteins are co-localized. Scale bar, 20 µm. \u003cstrong\u003eF\u003c/strong\u003e The co-localizations of Tim-4 and Axl or Tyro3 in Fig. 1E were quantified using ImageJ. Data are mean ± s.e.m. from \u003cem\u003en\u003c/em\u003e = 22 for Axl + Tim-4 and \u003cem\u003en\u003c/em\u003e = 28 for Tyro3 + Tim-4 randomly acquired images. ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; two-tailed unpaired Student’s \u003cem\u003et\u003c/em\u003e test.\u003c/p\u003e","description":"","filename":"Figure1250831.png","url":"https://assets-eu.researchsquare.com/files/rs-7580891/v1/10c3a989639c83c94c00ecca.png"},{"id":93665843,"identity":"d81b5078-d6f3-4a48-8cb0-586bceb3a1a8","added_by":"auto","created_at":"2025-10-16 09:03:39","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":570802,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe extracellular region of Axl binds to the IgV domain of Tim-4.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e The gene expression profile of TAM receptors and Tim-4 from the UniProt database. The relative expression of each gene is indicated by the color gradient. \u003cstrong\u003eB\u003c/strong\u003e. The truncated mutants of Axl and Tim-4. Ig, immunoglobulin; FnIII, fibronectin type-III; PKD, protein tyrosine kinase domain; IgV, Immunoglobulin variable; ECR, extracellular region. \u003cstrong\u003eC\u003c/strong\u003e-\u003cstrong\u003eF\u003c/strong\u003e 293T cells transfected with the indicated plasmids were lysed and proteins in lysates were precipitated with anti-HA (\u003cstrong\u003eC\u003c/strong\u003e), anti-FLAG (\u003cstrong\u003eD\u003c/strong\u003e, \u003cstrong\u003eF\u003c/strong\u003e), or anti-GFP (\u003cstrong\u003eE\u003c/strong\u003e) antibody-conjugated agarose beads. Bound proteins were detected with the indicated antibodies. An arrow and an arrow head in Fig. 2E indicate Tim-4\u003csup\u003emucin\u003c/sup\u003e and Tim-4\u003csup\u003eIgV\u003c/sup\u003e, respectively. IP, immunoprecipitation; TCL, total cell lysates.\u003c/p\u003e","description":"","filename":"Figure2250729.png","url":"https://assets-eu.researchsquare.com/files/rs-7580891/v1/19b40e93c8b1084260358cbf.png"},{"id":93665565,"identity":"8bbf3680-8997-482e-a163-426660631a9b","added_by":"auto","created_at":"2025-10-16 08:55:41","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":380629,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAxl facilitates Tim-4-mediated efferocytosis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA \u003c/strong\u003eLR73 cells stably expressing HA-Tim-4 were incubated with apoptotic thymocytes in serum-free medium for the indicated times, extensively washed with PBS to remove unengulfed apoptotic cells, and lysed. HA-Tim-4 was immunoprecipitated with anti-HA antibody-conjugated agarose beads. Co-immunoprecipitated endogenous Axl was detected with an anti-Axl antibody. \u003cstrong\u003eB\u003c/strong\u003e, \u003cstrong\u003eC\u003c/strong\u003e LR73 cells transfected with the indicated plasmids and GFP were incubated with pHrodo-labeled apoptotic thymocytes in serum-free medium for 2 h, washed with PBS, trypsinized, and analyzed using flow cytometry. Double-positive cells for pHrodo and GFP were considered to be phagocytes engulfing apoptotic cells (\u003cstrong\u003eB\u003c/strong\u003e) and the mean fluorescence intensity (MFI) of engulfing phagocytes was measured (\u003cstrong\u003eC\u003c/strong\u003e). Data are mean ± s.e.m. from \u003cem\u003en\u003c/em\u003e = 5 independent experiments. NS, not significant; *P \u0026lt; 0.05; ***P \u0026lt; 0.001; one-way ANOVA.\u0026nbsp; \u003cstrong\u003eD\u003c/strong\u003e LR73 cells transfected with Tim-4 alone or Tim-4 and Axl were incubated with TAMRA-labeled apoptotic thymocytes in serum-free medium at 4 \u003csup\u003eo\u003c/sup\u003eC for 2 h, extensively washed with PBS, fixed, and stained with anti-HA and anti-Axl antibodies. Bound apoptotic thymocytes were observed by confocal microscopy (left) and quantified (right). Arrow heads indicate bound apoptotic cells. Scale bar, 20 µm. Data are mean ± s.e.m. from \u003cem\u003en\u003c/em\u003e = 39 for Tim-4 and \u003cem\u003en\u003c/em\u003e = 41 for Tim-4 + Axl randomly acquired images. NS, not significant; two-tailed unpaired Student’s t test.\u003c/p\u003e","description":"","filename":"Figure3250831.png","url":"https://assets-eu.researchsquare.com/files/rs-7580891/v1/3449723913378640c8cdb03b.png"},{"id":93665548,"identity":"d7836a8d-44db-487f-9b5a-0890ee0e7eab","added_by":"auto","created_at":"2025-10-16 08:55:39","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":199073,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAxl signaling is necessary for the synergistic effect of Axl on Tim-4-mediated efferocytosis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e 293T cells transfected with the indicated plasmids were lysed and GST-Axl\u003csup\u003eECR\u003c/sup\u003e was precipitated with glutathione Sepharose beads in the presence or absence of purified Mertk\u003csup\u003eFnIII\u003c/sup\u003e. Co-precipitated proteins were detected with the indicated antibodies. TCL, total cell lysates; ECR, extracellular region. \u003cstrong\u003eB\u003c/strong\u003e LR73 cells transfected with the indicated plasmids and GFP were incubated with pHrodo-stained apoptotic thymocytes in serum-free medium in the presence or absence of purified Mertk\u003csup\u003eFnIII\u003c/sup\u003e for 2 h and analyzed using flow cytometry. Data are mean ± s.e.m. from \u003cem\u003en\u003c/em\u003e = 3 independent experiments. NS, not significant; *P \u0026lt; 0.05; two-way ANOVA. \u003cstrong\u003eC\u003c/strong\u003e LR73 cells transfected with the indicated plasmids and GFP were incubated with pHrodo-stained apoptotic thymocytes in serum-free medium for 2 h and analyzed using flow cytometry. Data are mean ± s.e.m. from \u003cem\u003en\u003c/em\u003e = 3 independent experiments. CT, cytoplasmic tail; NS, not significant; ***P \u0026lt; 0.001; one-way ANOVA. \u003cstrong\u003eD\u003c/strong\u003e, \u003cstrong\u003eE\u003c/strong\u003e LR73 cells transfected with the indicated plasmids and GFP were pre-incubated with R428 (\u003cstrong\u003eD\u003c/strong\u003e) or LY294002 (\u003cstrong\u003eE\u003c/strong\u003e) for 20 min. Then the cells were incubated with pHrodo-stained apoptotic thymocytes in serum-free medium for 2 h and analyzed using flow cytometry. Data are mean ± s.e.m. from \u003cem\u003en\u003c/em\u003e = 3 independent experiments. NS, not significant; ***P \u0026lt; 0.001; two-way ANOVA.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure4250831.png","url":"https://assets-eu.researchsquare.com/files/rs-7580891/v1/4086fcaf6b9252c360a0ad42.png"},{"id":93665564,"identity":"51d4d2f0-9f5e-47ed-aec4-7ed3a87c1a13","added_by":"auto","created_at":"2025-10-16 08:55:41","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":347561,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAxl deficiency impairs efferocytosis by splenic macrophages\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e The gene expressions of known PS receptors in the spleen were generated using single cell RNA sequencing data from the Expression Atlas database. The notation at the bottom of the heatmap indicates metadata file names. \u003cstrong\u003eB\u003c/strong\u003e Splenic macrophages were stained with anti-Tyro3, anti-Axl, or anti-Mertk antibodies and analyzed using flow cytometry. The dotted line in the histogram indicates the peak of the fluorescence intensity of control cells. \u003cstrong\u003eC\u003c/strong\u003e Splenic macrophages derived from \u003cem\u003eWT\u003c/em\u003e or \u003cem\u003eAxl\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice were incubated with TAMRA-stained apoptotic thymocytes in serum-free medium at 4 \u003csup\u003eo\u003c/sup\u003eC for 30 min, washed with PBS, fixed, stained with an anti-F4/80 antibody, and observed by confocal microscopy (left). Bound apoptotic thymocytes were quantified (right). Scale bar, 20 µm. Data are mean ± s.e.m. from \u003cem\u003en\u003c/em\u003e = 22 for \u003cem\u003eW\u003c/em\u003eT and \u003cem\u003en\u003c/em\u003e = 20 for \u003cem\u003eAxl\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e randomly acquired images. NS, not significant; two-tailed unpaired Student’s t test. \u003cstrong\u003eD\u003c/strong\u003e Splenic macrophages derived from the indicated mice were incubated with pHrodo-stained apoptotic thymocytes in serum-free medium for 30 min and analyzed using flow cytometry. Data are mean ± s.e.m. from \u003cem\u003en\u003c/em\u003e = 3 independent experiments. **P \u0026lt; 0.01; two-tailed unpaired Student’s t test. \u0026nbsp;\u003cstrong\u003eE\u003c/strong\u003e Splenic macrophages from \u003cem\u003eWT\u003c/em\u003e and \u003cem\u003eAxl\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice were pre-treated with R428 for 20 min and then incubated with pHrodo-stained apoptotic thymocytes in serum-free medium for 30 min. Engulfing phagocytes were analyzed using flow cytometry. Data are mean ± s.e.m. from \u003cem\u003en\u003c/em\u003e = 3 independent experiments.\u003c/p\u003e","description":"","filename":"Figure5250831.png","url":"https://assets-eu.researchsquare.com/files/rs-7580891/v1/6216d9df772e6a95df8abe7b.png"},{"id":98774572,"identity":"9fef905e-196d-4f2b-93f3-7faa1d3335f1","added_by":"auto","created_at":"2025-12-22 12:01:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2645988,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7580891/v1/a781dbd9-6091-4b81-a1b5-07fae7fc7922.pdf"},{"id":93665549,"identity":"f43a39c6-520a-44bd-8a2a-bbb18d09d6da","added_by":"auto","created_at":"2025-10-16 08:55:39","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2338039,"visible":true,"origin":"","legend":"Supplementary Figures","description":"","filename":"SupplementaryFigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-7580891/v1/ad138619edbc817c69170418.docx"}],"financialInterests":"(Not answered)","formattedTitle":"Tim-4 internalizes apoptotic cells through Axl-mediated signal transduction","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEfferocytosis, phagocytosis of apoptotic cells, is an essential process for both development and homeostasis [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. A hallmark of this process is the ability of phagocytes to recognize apoptotic cells, distinguishing them from viable cells. The ability is mediated by specific molecules expressed on apoptotic cells and phagocytes. Apoptotic cells present unique molecular markers on their surface that differentiate them from live cells, while phagocytes express corresponding receptors. One of the best-known ligands on apoptotic cells is phosphatidylserine (PS), which is normally confined to the inner leaflet of the plasma membrane in live cells but becomes exposed on the outer surface during apoptosis [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe exposed PS is recognized by phagocytes either directly or indirectly. Receptors capable of direct PS recognition, known as PS receptors, include BAI1, Tim-4, Tim-1, and Stabilin-2 [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In contrast, TAM receptor tyrosine kinases (Tyro3, Axl, and Mertk) and integrins bind to PS indirectly through bridging molecules such as Gas6 and Mfge8 [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Among PS receptors, Tim-4, also known as T-cell immunoglobulin and mucin domain-containing protein 4 (Timd4), has attracted significant attention in the field of efferocytosis due to its specific expression in macrophages and the notable impairment in efferocytosis observed upon its loss [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Originally, Tim-4 was identified as a Tim-1-interacting protein with a role in T-cell proliferation [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Later, a screening of antibodies against mouse peritoneal macrophages revealed that Tim-4 functions as a PS-binding protein [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Subsequent studies on Tim-4 as a PS receptor elucidated the structure of the Tim-4\u0026ndash;PS complex and highlighted its pivotal roles in efferocytosis and autoimmune diseases [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. However, the downstream signaling pathways of Tim-4 remain largely unexplored. Some studies have suggested potential downstream signaling mechanisms, but it is generally accepted that Tim-4 itself is unable to directly transduce signals into phagocytes [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Given that Tim-4 lacks direct signaling, it relies on a co-receptor, biochemically interacting with Tim-4, to transduce signals into phagocytes. Alternatively, Tim-4 functions solely as a tethering receptor, securing apoptotic cells to facilitate their engulfment by another receptor. These two mechanisms are not mutually exclusive and may coexist. Mertk, a member of TAM receptor family, exemplifies the former mechanism, whereas integrins represent the latter [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe TAM receptor family is a unique group of receptor tyrosine kinases involved in various cellular processes such as cell migration, survival, and efferocytosis [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. In the context of efferocytosis, TAM receptors bind to PS on apoptotic cells via bridging molecules such as Gas6 and Protein S, which induce dimerization of TAM receptors and facilitate the connection between apoptotic cells and TAM receptors. Both Gas6 and Protein S share a similar multidomain structure, where their SHBG domain interacts with the Ig like domains of TAM receptors and their Gla domain binds to PS. Gas6 can bind to all TAM family members, whereas Protein S preferentially binds to Mertk and Tyro3, with minimal affinity for Axl. Unlike Tim-4, TAM receptors can independently transmit signals for phagocytosis of the apoptotic cells they recognize [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eInterestingly, our previous research demonstrated that Mertk biochemically interacts with Tim-4 through an interaction between the FnIII domain of Mertk and the IgV domain of Tim-4, thereby promoting Tim-4-mediated efferocytosis [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. In this study, we investigated whether the other TAM receptors also biochemically interact with Tim-4 and, if any, play a cooperative role for Tim-4-mediated efferocytosis. Our results demonstrate that all TAM receptors interact with Tim-4, with Axl exhibiting the strongest interaction. The IgV domain of Tim-4 is responsible for its interaction with Axl, while Axl interacts with Tim-4 via both its FnIII and Ig like domains. Functionally, although Axl expression alone failed to promote efferocytosis, its co-expression with Tim-4 synergistically enhanced efferocytosis under serum-free conditions. Additionally, disrupting Tim-4\u0026ndash;Axl interaction or blocking Axl signaling abolished the synergistic effect of Axl on Tim-4-mediated efferocytosis. Taken together, our findings suggest that Axl functions as a co-receptor, transducing signals generated by Tim-4 into phagocytes to induce apoptotic cell internalization through its biochemical interaction with Tim-4.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eCell culture and transfection\u003c/h2\u003e\u003cp\u003eHEK 293T cells were cultured and maintained in DMEM (Dulbecco\u0026rsquo;s Modified Eagle\u0026rsquo;s Medium) containing 10% FBS (Fetal Bovine Serum) and 1% PSQ (Penicillin-Streptomycin-Glutamine). LR73 CHO cells were cultured in α-MEM (Alpha\u0026rsquo;s Modified Eagle\u0026rsquo;s Medium) supplemented with 10% FBS and 1% PSQ. 293T and LR73 cells were transfected using CaPO\u003csub\u003e4\u003c/sub\u003e (following the lab\u0026rsquo;s protocol) and Lipofectamine 2000 (Invitrogen, Waltham, MA, USA) respectively. To obtain primary splenic macrophages, the spleens of C57BL/6 mice were collected and minced with a razor blade into 1mm\u003csup\u003e3\u003c/sup\u003e pieces and transferred to a culture dish. Thereafter, the medium was changed every two days to remove stromal cells and red blood cells. After one week, the adherent cells were detached using trypsin-EDTA and used for experiments.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eMice\u003c/h3\u003e\n\u003cp\u003eC57BL/6 mice were purchased from Taconic Biosciences (Germantown, NY, USA). \u003cem\u003eAxl\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice were gifted from Prof. Hyung-Sik Kang at Chonnam National University (Republic of Korea). 3\u0026thinsp;~\u0026thinsp;4-week-old mice were used to prepare apoptotic thymocytes. 8\u0026thinsp;~\u0026thinsp;10-week-old mice were used to obtain spleen macrophage. All mice were housed in a well-maintained animal facility under controlled temperature and humidity conditions. All mouse-related procedures were reviewed and approved by the Institutional Animal Care and Use Committee of the Gwangju Institute of Science and Technology, following the guidelines outlined by the National Institutes of Health for the ethical treatment and use of laboratory animals.\u003c/p\u003e\n\u003ch3\u003ePlasmids and reagents\u003c/h3\u003e\n\u003cp\u003eAll plasmids in this study were constructed and sequenced to confirm their identity. Tim-4\u003csup\u003eECR\u003c/sup\u003e, Tim-4\u003csup\u003eIgV\u003c/sup\u003e and Tim-4\u003csup\u003eMucin\u003c/sup\u003e plasmids used in this study have been previously described [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Axl\u003csup\u003eECR\u003c/sup\u003e, extracellular region of Axl (residues 19\u0026ndash;445), Axl\u003csup\u003eIg\u003c/sup\u003e, immunoglobulin domain (residues 19\u0026ndash;217), and Axl\u003csup\u003eFnIII\u003c/sup\u003e, fibronectin type III domain (residues 218\u0026ndash;448) were cloned into the pEBG-GST vector or pEBB-GFP vector using PCR-based methods. The inhibitors and other reagents used in this study were R428 (S2841; Selleckchem, Huston, TX, USA), LY294002 (70920; Cayman Chemical Company), TAMRA-SE (C1171; Invitrogen, Carlsbad, MA, USA) pHrodo Red-SE (P36600; Invitrogen) Dexamethasone (D1756; Sigma-Aldrich, St. Louis, MO, USA). The antibodies used in the study were anti-Axl (AF854; R\u0026amp;D systems, Minneapolis, MN, USA), anti-Tyro3 (AF759; R\u0026amp;D systems), anti-Mertk (AF591, R\u0026amp;D Systems), anti-Tim-4 PE conjugated (12-5866-80; Invitrogen), anti-mouse F4/80 FITC clone:BM8 (123108, Biolegend, CA, USA) anti-FLAG (F1804, Sigma Aldrich, MO, USA), anti-HA (SC-7392, Santa Cruz biotechnology, Dallas, TX, USA), anti-HA (#3724, Cell signaling technology, Danvers, MA, USA), anti-GFP (ab290, Abcam, Cambridge, MA, USA), anti-GST (SC-138, Santa Cruz biotechnology) and normal goat IgG control (AB-108-C, R\u0026amp;D Systems). Fluorochrome-conjugated donkey anti-goat (Alexa Fluor 488, A-11055) and goat anti-rabbit secondary antibodies (Alexa Fluor 594, A-11037, Alexa Fluor 405, A-31556) were purchased from Invitrogen.\u003c/p\u003e\n\u003ch3\u003eBinding assay\u003c/h3\u003e\n\u003cp\u003eLR73 cells were plated on poly-D-lysine coated 18mm cover glass in a 12-well nontreated culture plate. One day after plating, the cells were transfected. The following day, cells were preincubated in serum-free alpha-MEM for 2 h and then incubated with 5-(and-6)-Carboxytetramethylrhodamine (TAMRA)-labelled apoptotic thymocytes suspended in serum free alpha-MEM at 4 \u003csup\u003eo\u003c/sup\u003eC for 2 h. After incubation, cells were washed with PBS, fixed and stained with PE-conjugated anti-Tim-4 and anti-Axl antibodies. Then cells were washed with PBS twice for 5 min each and stained with an Alexa fluor 488-conjugated secondary antibody for 1 h. Images were acquired by confocal microscopy (FV3000; Olympus, Tokyo, Japan). Spleen macrophages were plated on confocal dish. The day after seeding, cells were incubated with TAMRA-labeled apoptotic thymocytes in the same manner as previously described for use in the binding assay [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Then cells were washed with PBS, fixed and stained with a FITC-conjugated anti-F4/80 antibody at 4 \u003csup\u003eo\u003c/sup\u003eC for 1 day. After staining, cells were washed with PBS twice and mounted. Images were acquired by confocal microscopy (FV3000).\u003c/p\u003e\n\u003ch3\u003eEfferocytosis assay\u003c/h3\u003e\n\u003cp\u003eEfferocytosis assays were performed as previously described [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Briefly, LR73 cells were plated in a 24-well culture plate and transfected with the plasmids. One day after transfection, the cells were pre-incubated in serum-free alpha-MEM for 2 h and incubated with TAMRA-stained apoptotic thymocytes in serum-free alpha MEM in the presence or absence of purified proteins and inhibitors (LY294002, R428) for 2 h. The cells were washed with ice-cold PBS, trypsinized, analyzed using flow cytometry (BD FACS Canto II). Data were analyzed by FLOWJO software (FlowJo LLC, OR, USA). Spleen macrophages derived from indicated mice were pre-incubated in serum-free DMEM for 2 h and incubated with pHrodo-labeled apoptotic thymocytes in serum-free DMEM for 1 h. The macrophages were washed with ice-cold PBS, trypsinized and analyzed using flow cytometry.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eImmunofluorescence microscopy\u003c/h2\u003e\u003cp\u003eLR73 cells were plated on poly-D-lysine-coated 18mm cover glasses in a 12-well nontreated culture plate and transfected with indicated plasmids. The day after transfection, cells were fixed with 4% paraformaldehyde in PBS and blocked with 3% bovine serum albumin. Then, cells were immunostained with anti-Axl, anti-Tyro3, PE conjugated anti-Tim-4 antibodies at 4 \u003csup\u003eo\u003c/sup\u003eC for 1 day. After washing with PBS, cells were stained with an Alexa Fluor 488-conjugated anti-goat secondary antibody. Then cells were washed with PBS and mounted on slide glass using Duolink mounting media with DAPI. Images were obtained using a confocal microscope (FV3000).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eImmunoprecipitation and Immunoblotting\u003c/h3\u003e\n\u003cp\u003e293T cells were plated on 100 mm cell culture dish and transfected with indicated plasmids using CaPO\u003csub\u003e4\u003c/sub\u003e. Then cells were lysed using lysis buffer (10 \u0026micro;g/mL AEBSF, 10 \u0026micro;g/mL aprotinin, 10 \u0026micro;g/mL pepstatin, 10 \u0026micro;g/mL leupeptin, 150 mM NaCl, 10 mM NaF, 10 mM NaPP, 1 mM Na3VO4, 1% Triton X-100, and 50 mM Tris (pH 7.6)). The lysates were incubated with indicated antibodies with Protein A/G-conjugated beads (10001D, 10003D, Thermo Fisher Scientific), FLAG-conjugated beads (A2220, Sigma Aldrich), or glutathione agarose beads (17-0756-01, GE healthcare, IL, USA). Bound proteins were loaded and separated on SDS-PAGE, transferred onto nitrocellulose membrane, and detected with appropriate antibodies.\u003c/p\u003e\n\u003ch3\u003eBioinformatics\u003c/h3\u003e\n\u003cp\u003eThe TAM receptors and Tim-4 expression profile in mouse tissues were retrieved from the UniProt database and visualized as a heatmap using GraphPad Prism 9. The expression of phosphatidylserine receptors in the mouse spleen was retrieved from the Expression Atlas database (EMBL-EBI). Metadata files, GSE41637 [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], GSE74747 [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], ERP000591 [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], and DRP001032 [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], were used to generate the expression heatmap of PS receptors.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eCo-localization index\u003c/h2\u003e\u003cp\u003eThe Image of Axl or Tyro3 and Tim-4 expressing cells were obtained using a confocal microscope. The co-localization index was assessed by measuring the overlap between red fluorescence (Tim-4) and green fluorescence (Axl or Tyro3) using ImageJ 1.4. A color threshold was applied to define yellow pixels representing co-localized signals, as described in a previous study [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], and the co-localization index was calculated as the proportion of yellow pixels relative to the total number of pixels within the cellular area.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eAll data are shown as the mean value\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Each experiment was performed independently at least three times. Statistical significance was analyzed by the Student\u0026rsquo;s two-tailed t-test for experiments involving two groups. A one-way analysis of variance (One-Way ANOVA) was performed for experiments involving three or more samples using GraphPad Prism 9 software (Prism 9, Graphpad Software, La Jolla, CA, USA). The significance was accepted when p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eThe members of the TAM receptor family interact with Tim-4\u003c/h2\u003e\u003cp\u003eTim-4 is known to promote efferocytosis by cooperating with various proteins [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. In a previous study, we reported that Tim-4 facilitates efferocytosis through biochemical interactions with proteins containing a FnIII domain [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Their FnIII domains bind to the IgV domain of Tim-4, mediating interaction with Tim-4 and leading to enhanced Tim-4-mediated efferocytosis. Interestingly, TAM receptors share high structural similarity, and all contain Ig like and FnIII domains and a kinase domain in their extracellular and intracellular region, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Besides, all TAM receptors are known to be receptors for efferocytosis as well. Nevertheless, it is still unclear whether Tyro3 and Axl also cooperate with Tim-4 for efferocytosis.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo this end, we first investigated whether they biochemically associate with Tim-4. We overexpressed TAM receptors along with HA-Tim-4 in 293T cells and performed an immunoprecipitation assay. Hemagglutinin (HA)-tagged Tim-4 co-immunoprecipitated with all TAM receptor family members when immunoprecipitated using an anti-FLAG antibody (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and Supplementary Fig.\u0026nbsp;1A). When a reverse immunoprecipitation assay was performed using an anti-HA antibody, both Axl or Tyro3 were still co-immunoprecipitated with HA-Tim-4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, D, and Supplementary Fig.\u0026nbsp;1B, C). Notably, Axl exhibited the strongest binding to Tim-4 while Mertk showed relative weak interaction with Tim-4 among the receptors. Next, to evaluate their interaction within cells, FLAG-tagged Axl or Tyro3 was co-expressed with HA-tagged Tim-4 in phagocytes, and their subcellular localization was analyzed using confocal microscopy. Axl and Tyro3, along with Tim-4, were found to be exclusively expressed along the cell boundary, indicating their localization in the plasma membrane. Noticeably, the fluorescence signal from Axl markedly overlapped with that from Tim-4, to a greater extent than from Tyro3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE and F). The data suggest that all TAM receptors biochemically interact with Tim-4 with varying affinities, especially implying a more potent cooperative role of Axl for Tim-4 in efferocytosis.\u003c/p\u003e\u003cp\u003e\u003cb\u003eThe IgV domain of Tim-4 mediates interaction with Axl.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn addition to the strong interaction between Axl and Tim-4, a robust correlation was observed between Tim-4 and Axl expression across multiple tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Given the greater relevance of Axl to Tim-4 compared to the other TAM receptors, we focused on Axl in subsequent studies concerning its relationship to Tim-4 in efferocytosis. Next, we investigated which regions of Axl and Tim-4 mediate their biochemical interaction. To this end, we generated various truncated mutants of Tim-4 and Axl to test whether the cytoplasmic tail or transmembrane domain of Tim-4 is required for the interaction (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Axl maintained its interaction with Tim-4-GPI, a glycosylphosphatidylinositol (GPI)-anchored Tim-4 mutant that lacks the cytoplasmic tail and transmembrane domain (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and Supplementary Fig.\u0026nbsp;2A), indicating that the interaction between Axl and Tim-4 is mediated through their extracellular regions (ECRs). We then performed an immunoprecipitation assay to verify that the ECRs of both proteins are sufficient to mediate the interaction and confirmed interaction between the ECR of Axl and the ECR of Tim-4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD and Supplementary Fig.\u0026nbsp;2B).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWe further examined which domain(s) of the ECRs of both proteins mediate the interaction. Tim-4 contains one IgV domain and one mucin domain in its extracellular region, while Axl possesses two Ig like domains and two FnIII domains in its extracellular region (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Immunoprecipitation assays showed that the IgV domain of Tim-4 co-immunoprecipitated with the ECR of Axl, whereas the mucin domain did not (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE and Supplementary Fig.\u0026nbsp;2C), indicating that the IgV domain of Tim-4 is necessary for the interaction. Unexpectedly, however, both the Ig like and FnIII domains of Axl co-immunoprecipitated with the Tim-4 ECR (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF and Supplementary Fig.\u0026nbsp;2D), which contrasts with the previously reported interaction between Tim-4 and Mertk, where only the FnIII domain of Mertk binds to Tim-4. These results indicate multiple binding sites on Axl for Tim-4, which may lead to a stronger interaction of Axl with Tim-4 than that of Mertk or Tyro3.\u003c/p\u003e\u003cp\u003e\u003cb\u003eTim-4 with Axl promotes efferocytosis synergistically.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eNext, we investigated the functional significance of the biochemical interaction between Axl and Tim-4 in efferocytosis. First, to determine whether Axl plays a role during Tim-4-mediated efferocytosis, we examined whether their biochemical interaction changes during efferocytosis. We co-incubated apoptotic cells with phagocytes overexpressing Tim-4 under serum-free conditions and assessed interaction of Tim-4 with endogenous Axl. Interestingly, without apoptotic cells, no detectable interaction between Tim-4 and endogenous Axl was observed. However, upon apoptotic cell stimulation, interaction between Tim-4 and endogenous Axl was induced (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and Supplementary Fig.\u0026nbsp;3A), implying that Axl is involved in Tim-4-mediated efferocytosis. To further confirm this, we overexpressed Tim-4 and Axl in phagocytes and performed an efferocytosis assay. We performed experiments under serum-free conditions without bridging molecules (e.g., Gas6) to ensure that Axl does not contribute to efferocytosis by itself, and serum-free conditions were used in all subsequent experiments. Despite the low basal efferocytosis level under serum-free conditions, phagocytes expressing Tim-4 exhibited significantly enhanced efferocytosis whereas the cells expressing Axl alone failed to promote efferocytosis compared to the control cells. Especially, phagocytes co-expressing Tim-4 and Axl showed far more efficient efferocytosis than those expressing Tim-4 alone, as measured by the percentage and MFI (mean fluorescence intensity, an indicator of the relative number of apoptotic cells per cell) of phagocytes engulfing apoptotic cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, C and Supplementary Fig.\u0026nbsp;4A). This efficient efferocytosis by phagocytes expressing both Tim-4 and Axl did not result from their altered ability to recognize apoptotic cells because the binding of apoptotic cells to phagocytes was comparable in cells expressing Tim-4 alone and those co-expressing Tim-4 and Axl (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD), implying Axl facilitates the internalization of apoptotic cell during Tim-4-mediated efferocytosis. Collectively, these data suggest that Axl transduces signals to promote the internalization of apoptotic cells secured by Tim-4 through the interaction with Tim-4 during efferocytosis.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eBlocking Axl signaling impairs Tim-4-mediated efferocytosis\u003c/h2\u003e\u003cp\u003eIf Axl is indeed a signal transducer for the internalization of apoptotic cells during Tim-4-mediated efferocytosis, then blocking the interaction or Axl signaling should impair Tim-4-mediated efferocytosis. In our previous study, we have shown that the FnIII domain of Mertk binds to the IgV domain of Tim-4 and its soluble form (Mertk\u003csup\u003eFnIII\u003c/sup\u003e) disrupts the interaction of Tim-4 with Mertk. Similarly, Mertk\u003csup\u003eFnIII\u003c/sup\u003e also abrogated the interaction between Axl and Tim-4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and Supplementary Fig.\u0026nbsp;5A). When phagocytes were treated with Mertk\u003csup\u003eFnIII\u003c/sup\u003e, efferocytosis was impaired to a comparable extent in both Tim-4-expressing phagocytes and Tim-4/Axl co-expressing phagocytes. However, efferocytosis remained unaffected in control cells or Axl-alone expressing cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), indicating that interaction of Axl with Tim-4 is necessary for the cooperative role of Axl for Tim-4-mediated efferocytosis. Next, to assess the effect of Axl signaling on Tim-4-mediated efferocytosis, we blocked Axl signaling using two different approaches. First, we generated a truncated mutant of Axl lacking its cytoplasmic tail (Axl\u003csup\u003eDCT\u003c/sup\u003e), unable to mediate intracellular signal transduction, and tested its effect on Tim-4-mediated efferocytosis. Efferocytosis by phagocytes co-expressing Axl\u003csup\u003eDCT\u003c/sup\u003e and Tim-4 was not enhanced and comparable to that by phagocytes expressing Tim-4 alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). As an alternative second approach to impair Axl signaling, we used R428 or LY294002, which specifically inhibits Axl or an Axl downstream molecule, PI3K, respectively. When phagocytes were treated with the inhibitors, efferocytosis by phagocytes expressing Tim-4 alone or Tim-4 and Axl was notably inhibited to a degree that was indistinguishable between the two groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD and E). These data indicate that Axl serves as a receptor that relays signals into phagocytes to promote the internalization of apoptotic cells secured by Tim-4 during efferocytosis.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eTim-4-mediated efferocytosis by splenic macrophages requires Axl.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe spleen is an organ where old or damaged red blood cells from the circulation are removed by splenic macrophages. We confirmed the high expression of Tim-4 and the dominant expression of Axl among TAM receptors in the spleen (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). In addition, of all the PS receptors, Tim-4 was found to be predominantly expressed in the spleen, supported by publicly available gene expression databases (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Specifically, splenic macrophages showed high Axl expression, relatively low Mertk expression, and undetectable Tyro3 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Therefore, to further validate the synergistic role of Axl for Tim-4-mediated efferocytosis in a more physiologically relevant system, we used splenic macrophages. We first evaluated the ability of wild-type (\u003cem\u003eWT\u003c/em\u003e) and Axl-deficient (\u003cem\u003eAxl\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e) splenic macrophages to recognize apoptotic cells. \u003cem\u003eWT\u003c/em\u003e and \u003cem\u003eAxl\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e splenic macrophages did not show a difference in their ability to bind to apoptotic cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). However, efferocytosis by \u003cem\u003eAxl\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e splenic macrophages was significantly less efficient than \u003cem\u003eWT\u003c/em\u003e splenic macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Interestingly, this superior efferocytosis by \u003cem\u003eWT\u003c/em\u003e splenic macrophages to \u003cem\u003eAxl\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e splenic macrophages was abolished when treated with an Axl specific inhibitor, resulting in indistinguishable efferocytosis between \u003cem\u003eWT\u003c/em\u003e and \u003cem\u003eAxl\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e splenic macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). These results imply that Axl transduces signals for the internalization of apoptotic cells secured by Tim-4 on splenic macrophages. In summary, our findings demonstrate that Axl serves as a signal transducer for Tim-4 in efferocytosis, promoting the internalization of apoptotic cells secured by Tim-4 through its interaction with Tim-4.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eWhen Tim-4 was first reported as a phosphatidylserine (PS) receptor recognizing PS during efferocytosis, its downstream signaling remained unclear. Subsequent studies demonstrated that even a truncated mutant of Tim-4 lacking a cytoplasmic tail could promote efferocytosis, leading to the classification of Tim-4 as a tethering receptor that cannot directly transmit signals [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Several studies have since proposed a two-step engulfment process, in which Tim-4 facilitates efferocytosis by coordinating with other engulfment receptors such as integrins [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Additionally, a co-receptor capable of biochemically interacting with Tim-4 to mediate signal transduction has been identified, significantly advancing our understanding of Tim-4 downstream signaling [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn this study, we investigated the biochemical interactions and functional roles of other TAM receptor family members that are structurally and functionally similar to Mertk, a previously reported binding partner of Tim-4. As expected, Axl and Tyro3, which share a high structural similarity with Mertk, both interacted with Tim-4. We found that the IgV domain of Tim-4 and the FnIII domain of Axl mediate the interaction. While the Mertk-Tim-4 interaction involves only the FnIII domain of Mertk binding to Tim-4, in the case of Axl, both its Ig like and FnIII domains bind to the extracellular region of Tim-4. This may contribute to Axl forming the strongest interaction with Tim-4 among TAM receptors. Additionally, given that the Ig-like domain of Axl is structurally and sequentially similar to the IgV domain of Tim-4, the Ig like domain of Axl can interact with its own FnIII domain, providing insights into the inhibition-activation mechanism of Axl. Further research on this interaction could be valuable in the future.\u003c/p\u003e\u003cp\u003eAll TAM receptor family members have been implicated in efferocytosis [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. However, their expression patterns differ: while Mertk and Axl exhibit similar expression patterns to Tim-4, Tyro3 shows distinct differences. Specifically, in many resident macrophages, Tim-4, Axl, and Mertk are expressed, whereas Tyro3 is not. Despite confirming the biochemical interaction between Tim-4 and Tyro3 in this study, the physiological role of Tyro3 in Tim-4-mediated cellular events remains unclear and warrants further investigation.\u003c/p\u003e\u003cp\u003eDuring TAM receptor-mediated efferocytosis, TAM receptors recognize apoptotic cells via Gas6 or ProS, leading to the activation of the PI3K-AKT signaling pathway [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. This activation subsequently induces Rac activation and cytoskeletal rearrangement, allowing apoptotic cells to be internalized [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Therefore, in Tim-4-mediated efferocytosis, apoptotic cells recognized by Tim-4 appear to be internalized through the activation of the PI3K-AKT signaling pathway via a biochemical interaction with Axl. In this study, we found that apoptotic cell stimulation under serum-free conditions induces the biochemical interaction between Tim-4 and Axl, suggesting that this interaction may contribute to Axl activation. The interaction between the FnIII domains and Ig like domains may act as a self-inhibitory mechanism for Axl. During Tim-4-mediated efferocytosis, this interaction-mediated self-inhibition of Axl could be relieved and activated upon Tim-4 binding to the domains of Axl. Investigating how the interaction between the Ig like domains and FnIII domains of Axl affects its activation could be an intriguing topic for future research.\u003c/p\u003e\u003cp\u003eIn conclusion, this study demonstrates that Axl synergistically promotes Tim-4-mediated efferocytosis by transducing apoptotic cell recognition signals into phagocytes through biochemical interaction with Tim-4, effectively substituting for the inability of Tim-4 to transduce signals into phagocytes. Given that both Tim-4 and Axl are associated with various immune disorders, our findings provide valuable insights for the development of therapeutic strategies targeting related diseases.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eConflict of Interest\u003c/h2\u003e\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\u003ch2\u003eAuthor contributions\u003c/h2\u003e\u003cp\u003eConceptualization, B.M. and D.P.; Methodology, B.M., J.J., E.O., J.H., J.L., E.H., C.L., and D.J.; Formal analysis, B.M., J.J., E.O., J.H., J.L., and D.P.; Resources, B.M. and J.J.; Writing, B.M. and D.P.; Funding acquisition, B.M. and D.P.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e\u003cp\u003eThis work was supported by the National Research Foundation of Korea (NRF) grant funded by the Ministry of Science and ICT and Ministry of Health \u0026amp; Welfare, Republic of Korea (No. RS-2025-00558157, RS-2024-00466906, and RS-2024-00510912) and by GIST research Institute (GRI) IIBR. The confocal imaging was performed using instruments provided by the GIST Advanced Institute of Instrumental Analysis (GAIA). We sincerely thank Prof. Hyung-Sik Kang at Chonnam National University for the generous gift of \u003cem\u003eAxl\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice.\u003c/p\u003e\u003ch2\u003eAvailability of Data and Materials\u003c/h2\u003e\u003cp\u003eFurther information and requests for materials should be directed to and will be fulfilled by the lead contact, Daeho Park ([email protected]). Plasmids generated in this study will be distributed upon request to other investigator under a Material Transfer Agreement.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eArandjelovic S, Ravichandran KS. 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Among them, Tim-4 has been studied to elucidate signaling since its identification. However, how apoptotic cells secured by Tim-4 are internalized remains still unclear. Here, we reveal that Axl-mediated signal transduction is necessary for Tim-4 to ingest apoptotic cells. We found correlated expression patterns for Tim-4 and Axl across various tissues, and biochemical and cell biological analyses showed that Tim-4 interacted most strongly with Axl among the TAM receptor family. Mechanistically, the immunoglobulin variable (IgV) domain of Tim-4 interacted with two distinct domains within the extracellular region of Axl. Co-expression of Tim-4 and Axl synergistically promoted Tim-4-mediated efferocytosis. Crucially, disrupting the interaction or pharmacologically or genetically blocking Axl signaling abolished the synergistic effect of Axl on Tim-4-mediated efferocytosis. These findings collectively demonstrate that during Tim-4-mediated efferocytosis, Axl acts as a key signaling relay, biochemically interacting with Tim-4 to transmit apoptotic cell recognition signals into phagocytes, thereby enabling efficient apoptotic cell ingestion.\u003c/p\u003e","manuscriptTitle":"Tim-4 internalizes apoptotic cells through Axl-mediated signal transduction","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-16 08:55:33","doi":"10.21203/rs.3.rs-7580891/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"cc36b7d6-4698-44f2-8456-cc2cc08e0ec8","owner":[],"postedDate":"October 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":55731080,"name":"Biological sciences/Cell biology/Cell death/Apoptosis"},{"id":55731081,"name":"Biological sciences/Cell biology/Cell signalling"}],"tags":[],"updatedAt":"2025-12-11T16:06:50+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-16 08:55:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7580891","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7580891","identity":"rs-7580891","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}