The secreted Nimrod protein NimB1 negatively regulates early steps of apoptotic cell phagocytosis in Drosophila

  1. Asya Dolgikh
  2. Samuel Rommelaere
  3. Aseel Ghanem
  4. Bianca Petrignani
  5. Mickael Poidevin
  6. Estee Kurant
  7. Bruno Lemaitre

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Abstract

Efferocytosis, the efficient clearance of apoptotic cells (ACs) by phagocytes, is vital for maintaining tissue homeostasis. Here, we reveal the role of the secreted protein NimB1 in reducing apoptotic cell recognition and binding in the early stages of efferocytosis. NimB1 is expressed in macrophages (also called plasmatocytes) and binds to ACs in a phosphatidylserine-dependent manner. Structural analysis shows that NimB1 shares striking similarities with the bridging molecule NimB4, and possesses two phosphatidylserine-binding motifs, supporting its role in efferocytosis. Larval macrophages of NimB1-null Drosophila mutants display a hyper-phagocytic phenotype characterized by increased engulfment of ACs. Confocal imaging reveals that NimB1 specifically inhibits early steps in internalization of ACs, but does not impact phagosome maturation. We find that NimB1 is a secreted factor that negatively regulates efferocytosis, antagonizing the role of NimB4. Our study and the analogous opposing roles of Draper Isoforms II and I in efferocytosis suggest that a balance of negative and positive regulators allows optimization of the rate of apoptotic cell clearance by macrophages.

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  1. Version published to 10.1242/dev.204919
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    Reply to the reviewers

    Dear Editor,

    Thank you for reviewing our article. We are happy to see that the reviewers are positive on our manuscript. We have tried to address nearly all their comments. Find below a point-by-point answer.

    With best regards,

    Bruno Lemaitre and Asya Dolgikh

    Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    This work defines NimB1 protein as a PS binding bridging molecule but with a negative regulatory role in efferocytosis. Specifically, the authors demonstrate via a variety of genetic, cell biological, and other approaches that loss of NimB1 leads to Drosophila macrophages being more adherent to apoptotic targets and engulf them more robustly. The authors also nicely demonstrate that the function of NimB1 differs from NimB4, and the double mutant demonstrating PS-binding yet, distinct roles. Further, the authors show that NimB1 does not affect bacterial phagocytosis.

    Overall, this is a well-done study. The authors have already done a very thorough job addressing the key points and I congratulate the authors.

    My only minor comment is that the authors could try to make the comment better about whether or not such a 'negative regulatory' bridging molecules may exist in other species, and particularly mammals. If so, this is quite novel. The authors refer to CD47 but this is a membrane protein. The other minor comment is whether the authors ever tried express the PS binding domains as a fusion protein - this would provide a more direct evidence for the binding to PS (although the authors do competitive inhibition with Annexin V). This could be commented upon although testing this is not necessary if they have not already done so.

    We greatly appreciate the reviewer’s positive feedback. In the revised manuscript, we have now included a more detailed discussion of mammalian proteins with analogous roles, specifically referencing Draper isoforms (I and II), the CD300 receptor family, and surfactant proteins A and B (see page 16).

    Reviewer #1 (Significance (Required)):

    The identification of the negative regulator bridging protein NimB1 is novel and could be broadly interesting to those studying efferocytosis.

    Regarding the suggestion to overexpress just the putative PS-binding domain of NimB1, we agree this could strengthen the evidence for its PS-binding function. However, generating a new transgenic fly line would require significant additional time. Moreover, the presence of a PS-binding motif was also proposed in the recent study on Orion (Ji et al., 2023), which we have cited in our manuscript. The Orion binds PS through a conserved RRY motif. This motif is critical for Orion’s ability to directly interact with PS and facilitate its secretion. Mutagenesis experiments disrupting the RRY motif—specifically substituting arginine residues with alanines—abolished Orion’s PS-binding capacity, demonstrating the essential role of this sequence. Functional assays also validated that Orion competes with Annexin V, a well-established PS-binding protein, for access to PS-exposing surfaces (Ji et al., 2023).

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    Summary:

    In this study, Dolgikh and colleagues propose a first investigation about the role of the drosophila Nimrod protein NimB1. Although the role of several members of the family in phagocytosis has been explored, the function of Nimrod type B proteins is less addressed. Within silico analysis, they first see a strong similarity between NimB1 and NimB4. They show that NimB1 is primarily expressed in phagocytes, and as NimB4 can bind phosphatidylserines (PS), leading to a possible shared role in efferocytosis. Using transgenic and null drosophila models, the authors then compare the impact of NimB1 overexpression or deficiency. They compare the effects shown to NimB4 and Draper deficient lines, as these two proteins were previously shown to play a role in efferocytosis. They propose that NimB1 is a secreted protein that binds apoptotic cells. They show that NimB1 deficiency changes the adhesion properties of macrophages. The major finding is that NimB1 delays the early stages of efferocytosis, contrary to NimB4 and Draper that on the contrary facilitate efferocytosis. In contrast, the authors propose that NimB1 increases the formation of phagosomes.

    We appreciate the reviewer’s acknowledgment that our key discovery centered around NimB1 functioning as a negative regulator of efferocytosis. This finding highlights NimB1’s distinct role compared to NimB4 and Draper, which instead promote the process.

    Major comments:

    One of the major technical challenges here was to generate models to allow the detection of the protein in cellulo and in vivo. Although the results are convincing in transgenic lines NimB1 expression is driven by the endogenous promoter, one could still argue that the GFP tags would lead to changes in the localization of the protein.

    We understand the concern regarding potential localization changes introduced by GFP tags. However, in previous studies, the same fosmid construct was applied to NimB4-sGFP, and produced a distinctly different expression pattern—NimB4-sGFP expression was more pronounced and clearly present in the glial cells in the brain (Petrignani et al, 2021: Figure EV1A). The fact that the NimB1-sGFP and NimB4-sGFP fosmids localized to different tissues suggests that possible any mis-localization changes due to the GFP tag do not override localization properties intrinsic to the proteins.

    In line with the previous comment, to show that NimB1 is a secreted protein, the authors use an overexpression model. How to be sure, that overexpression itself does not lead to increased secretion, or shedding from the membrane?

    The observation that uas-NimB1-RFP accumulates in the nephrocytes upon Lpp-Gal4 (fat body) expression, and the presence of a signal peptide suggests that this protein can be secreted.

    We cannot exclude that in endogenous condition, NimB1, remains attached to hemocytes. We have confirmed that the Lpp driver is not expressed in nephrocytes.

    Would an experiment with a control consisting in a known protein secreted by macrophages lead to the same staining pattern (positive control)? Could another methodology like a Western Blot on supernatants from hemocyte cell culture (over)expressing NimB1, with an anti-RFP staining, be envisaged?

    We have already performed similar experiment with other secreted proteins such as NimB4-GFP (Petrignani et al., 2021: Figure: 1B). In the revised article, we have added Viking-RFP as a positive control of a secreted protein (Figure S1F). Figure S2 shows a Western blot with hemolymph extract. We detected NimB1-RFP at its expected molecular weight of 44 kDa, verifying that is present into the hemolymph (Supplementary Document S2 C).

    It sems counterintuitive that phagocytes from Draper and NimB4 null mutants with defects in efferocytosis show increased load of apoptotic cells (Figure 6C and D in both unchallenged and injury condition). Do the authors have precedent data to cite going to the same direction? Are cell debris engulfed but not degraded efficiently?

    The observation that Draper and NimB4 null mutants have an increased load of apoptotic cells has already been reported in the literature. Several studies have now shown that Draper is not always required for the initial uptake of apoptotic corpses but is critical for phagosome maturation (Meehan et al., 2016; Serizier et al., 2022; Serizier & McCall, 2017). In our article on NimB4 (Petrignani et al., 2021), we have previously shown that the accumulation of immature phagosomes that are not properly eliminated indirectly impairs the uptake of new apoptotic corpses. This explains why efferocytosis is then impaired only at late time points, when unresolved phagosomes have accumulated to the threshold that prevents further phagocytosis.

    In Figure 6D it seems indeed that NimB4, NimB1/NimB4 and Draper mutants do not accumulate more apoptotic material upon injury. However, levels for NimB4 is close to the one obtained with NimB1 mutants. Is it statistically true? If yes, what could be the reason for this similarity? In any case, as some important conclusion relies on the comparison between UC and injury conditions, adequate statistics and representations could be proposed.

    We thank the reviewer for this pertinent observation and the opportunity to clarify. In the unchallenged (UC) condition, NimB4sk2 and draperΔ5 mutants indeed exhibit significantly elevated levels of apoptotic cell (AC) content in macrophages compared to wild-type and NimB1 mutant genotypes (****p crimic* and NimB1229/NimB1crimic mutants show significantly lower levels in the UC condition, consistent with a role for NimB1 in early recognition or regulation of phagocytic initiation, not in corpse degradation.

    In contrast, upon injury (90 minutes post-challenge) we observe a statistically significant increase in apoptotic material in NimB1 mutants compared to UC hemocytes of the same genotype (****p sk2* and draperΔ5 mutants between the UC and 90 min conditions (ns for NimB4). This is consistent with their known defect in corpse degradation, which results in saturation of phagocytic capacity at baseline, and an inability to respond further upon challenge with apoptotic cells.

    While the absolute levels of apoptotic material in injured NimB1 and UC NimB4 mutants appear similar at first glance, statistical testing confirms that they are significantly different. NimB4 mutant macrophages retain apoptotic debris due to defective degradation, whereas NimB1 mutants have an increase in newly acquired apoptotic content due to enhanced uptake.

    Additionally, NimB161, NimB4sk2 double mutants display a partial increase in apoptotic load upon injury (****p To directly address the reviewer’s suggestion, we have now recalculated and visualized key comparisons with appropriate statistical testing, as shown in Revision Figure 1. All statistical analyses were conducted using unpaired two-tailed Student’s t-tests. This additional figure allows clearer evaluation of genotype-specific differences at both baseline and post-injury conditions and supports our conclusions that NimB1 and NimB4 regulate distinct stages of phagocytosis. We have also clarified the text to better explain that both NimB4 and Draper mutants accumulate unresolved apoptotic material under baseline conditions, and do not accumulate further material upon challenge, due to a block in phagosome maturation.

    Revisions Figure 1.

    __Quantification of phagocytic events in wild-type and mutant macrophages under unchallenged and post-injury conditions __

    (A) Comparison of phagocytic events per frame in w1118 (wild-type), NimB1crimic, NimB1229/NimB1crimic, NimB4sk2, NimB161,NimB4 sk2, and draperΔ5 larvae under unchallenged conditions (UC) and 90 minutes after injury (90 min). Data are presented as individual data points with means. Statistical significance was determined using Student's t-test (*P (B) Direct comparison of phagocytic events between NimB1crimic (red) and NimB4sk2 (gray), and between NimB1229/crimic (dark red) and NimB4sk2 (gray) under both unchallenged (UC) and post-injury (90 min) conditions.

    The authors claim with analyses of Figure 8C and D, that NimB1 mutants show acidic vehicles normal in size and fluorescence intensity. However, statistical differences are still observed compared to control condition, which is also seen in representative images shown.

    In Figure 8C and D, we provide two quantitative measures to clarify the size and intensity of acidic vesicles. First, we show that mean fluorescence in hemocytes is elevated for all NimB and draper mutants compared to wild type, indicating an overall increase in internalized material. However, we also quantified the number of vesicles per hemocyte and found that NimB1 mutants exhibit significantly more vesicles. Despite this increase, the representative images do not show an obvious enlargement of individual vesicles, suggesting that while more material is being taken up, the vesicles themselves are not enlarged. The enlarged vesicles in case of NimB4 or draper mutant would result from the unresolved cargo (Petrignani et al., 2021). This distinction underscores that higher fluorescence values reflect increased cargo internalization, rather than the larger vesicular structures that result from impaired degradation as in NimB4 or draper mutants.

    Minor comments:

    In figure 2D, what allows to say the expression is restricted in macrophages? Is it the colocalization with SIMU being a macrophage-specific marker?

    In Figure 2D, we relied on SIMU as a macrophage-specific marker in Drosophila embryos to determine that NimB1 expression is restricted to macrophages. Previous research has demonstrated that SIMU is predominantly expressed in embryonic macrophages (where it is essential for apoptotic cell clearance) (Kurant et al., 2008; Roddie et al., 2019). Consequently, the colocalization of NimB1 signal with SIMU-positive cells strongly indicates that NimB1 is confined to macrophages during this developmental stage.

    In figure S3B and C, it appears that double NimB1/NimB4 mutants exhibit less spreading than single ones (especially NimB4). Is it the case (statistical significance). If yes what could be the explanation?

    Yes, the double NimB1, NimB4 mutants exhibit higher number of hemocytes and significantly reduced cell spreading compared to single mutants. The phenotype is similar to NimC1, eater double mutants (Melcarne et al., 2019) which also show higher number of hemocytes, reduced cell spreading and also diminished capacity to phagocytose apoptotic cells (and, in the case of NimC1, Eater, bacteria as well) (Melcarne et al., 2019). A likely explanation lies in impaired membrane remodeling critical for pseudopod extension and phagosome formation. Studies have shown that defects in actin polymerization or membrane tension can hinder pseudopod extension, reducing phagocytic efficiency (Lee et al., 2007; Masters et al., 2013). Same for the decreased ability of these mutants to form filopodium, a process essential for effective target engagement and engulfment. Filopodia play a significant role in capturing particles and directing them toward the macrophage body for engulfment (Horsthemke et al., 2017). Disruptions in these pathways lead to reduced phagocytic efficiency and a more rounded macrophage morphology, as the cells fail to spread properly (Horsthemke et al., 2017; Lillico et al., 2018). Other than these general observations, we do not have an explanation as to why NimB1, NimB4 double mutants specifically show a higher number of hemocytes and reduced cell spreading.

    Several graphs are identical between figure 4 and S4. It is probably not useful and complicates reading.

    We agree that duplicating these graphs complicates the presentation. Therefore, we have removed the redundant graphs in the supplementary materials, ensuring the data are shown only once to maintain clarity and ease of reading

    As TEM images shown in Figure 8B do not lead to quantitative data, I would put it as supplementary file.

    We agree that the TEM images in Figure 8B do not provide strictly quantitative data. To streamline the main manuscript, we have relocated these images to the supplementary section in the revised version

    Reviewer #2 (Significance (Required)):

    This study uses several approaches and models to address the role of NimB1 in efferocytosis. Both In Vitro and In Vivo approaches are proposed. They give insight into the role of this protein with unknown function so far. Some statistical analysis could be performed to improve the clarity of conclusions. One of the important aspects is the secreted nature of NimB1.However, additional approaches could be proposed to confirm this.

    Basic immunologists and cell biologists would be interested in reading this article that highlights the delicate equilibrium between pro and anti-efferocytosis molecules.

    I am an immunologist/cell biologist with expertise in lysosomal catabolism. As I work on mouse models or Human samples, my mastering of drosophila as a model is limited.

    We thank the reviewer for the positive evaluation of our work. In this revision, we have added further detail to clarify the properties of NimB1 as a secreted protein and strengthen our data presentation. By providing additional clarity on methods and interpretations, we hope immunologists and cell biologists—including those who do not routinely work with Drosophila—will find our findings more accessible.

    Reviewer #3 (Evidence, reproducibility and clarity (Required)):

    This paper investigates the role of NimB1, a secreted member of the Nimrod family in Drosophila, in the process of efferocytosis, the clearance of apoptotic cells by macrophages. Previous studies have identified NimB4, another secreted Nimrod protein, as a positive regulator of efferocytosis, enhancing both apoptotic cell binding and phagosome maturation. In contrast, the authors propose that NimB1 functions as a negative regulator, slowing down the early stages of apoptotic cell binding and internalization. This regulatory balance is suggested to fine-tune efferocytosis to maintain homeostasis.

    The primary aim of this study was to characterize the function of NimB1 to better understand the roles of proteins within the NimB family.

    This study identifies a novel function for NimB1 in modulating the early stages of efferocytosis, adding to our understanding of how Nimrod proteins fine-tune apoptotic cell clearance. The authors establish a clear phenotypic contrast between NimB1 and NimB4, which provides a compelling framework for understanding how positive and negative regulators coordinate phagocytosis. It also highlights the multiple roles of the secreted members of the Nimrod scavenger receptor family, which have remained so far poorly investigated.

    This is an interesting study that could be strengthened by additional validation and broader experimental support. As the authors point out in the discussion, it is known that PS bridging molecules contribute to phagocytosis and that the contribution of positive and negative players finely tune phagocytosis in flies and mammals. Clarifying the mode of action of NimB1 in those processes would higher the impact of this interesting piece of work. For example, does NimB1 interact with NimB4 and if so, what is the role of this interaction? How does NimB1 integrate in the signaling cascade that allows scavenger receptors to bind PS? Does it act similar to Orion by enhancing the PS binding of a scavenger receptor?

    Key Findings • NimB1 and NimB4 are structurally similar, as supported by AlphaFold2 modeling, suggesting functional relatedness. • NimB1 is expressed in macrophages, secreted into the hemolymph, and binds apoptotic cells in a phosphatidylserine (PS)-dependent manner. • NimB1 is induced by challenge. • NimB1 mutants display a hyper-phagocytic phenotype, with faster recognition and internalization of apoptotic cells. • NimB1 loss enhances macrophage adhesion and actin remodeling, while bacterial phagocytosis remains unaffected, suggesting a specific role in apoptotic clearance. • NimB1 acts early in the phagocytic process, while NimB4 functions at later stages, particularly in phagosome maturation.

    We thank the reviewer for their positive assessment and are pleased that our findings identify NimB1 as a novel secreted negative regulator of efferocytosis, underscoring a greater level of regulatory complexity in apoptotic cell clearance.

    Unfortunately, attempts to produce functional NimB1 protein were not successful, limiting our ability to address some of the reviewer’s suggestions experimentally. Despite these challenges, the evidence we present—particularly from our genetic assays—clearly indicates that NimB1 exerts an inhibitory influence during the early steps of apoptotic cell binding, distinguishing it from the late-stage promoting function of NimB4.

    Major comments:

    Figure 1: AlphaFold is a valuable tool for generating hypotheses, however predicted structures should not be presented as definitive evidence of similarity, particularly without complementary experimental validation. This section would be stronger if the structural predictions were explicitly framed as predictions. In the absence of such data, the interpretation should be toned down.

    We agree with the reviewer and we have now framed our observation as prediction and toned down our interpretation. We also note that the similarities between NimB4 and NimB1 are also underlined by the phylogenetic analysis and expression pattern.

    Figure 2DE: Given its basal level in homeostatic conditions, it would have been useful to look at the NimB1-GFP upon challenge. Also, the authors show only a single larval macrophage with no comparison point. To strengthen this result, the authors could include another protein quantification method, such as western blotting. Alternatively, labelling of NimB1>UASmRFP in embryo that present the highest expression levels would also strengthen this result.

    Unfortunately, we cannot currently perform additional experiments on embryos within the scope of this project because those experiments were performed by our collaborators in Haifa (Estee Kurant Lab). Repeating them would require sending the lines to their lab and accommodating their experimental schedule and manpower constraints.

    In supplementary Figure S1F: the authors overexpress NimB1-RFP using the fat body driver Lpp-Gal4 and show larvae with RFP in the nephrocyte. Could filet preparations be shown? Could the authors present evidence that the Lpp driver is not expressed in the nephrocytes (or refer to literature)?

    The Lpp-Gal80 driver is described as fat body-specific and has been used to manipulate gene expression in the fat body in many other studies. We have checked Lpp-Gal80>UAS-GFP expression in larvae and did not observe expression in larval nephrocytes. The whole live larvae were observed under the microscope with no prior filet preparations. To provide the evidence that Lpp is not expressed in the nephrocytes we are providing the images of the whole larvae expressing GPF from the Lpp, as per genotype: Lgg-Gal80>UAS-GFP (see below, Revisions Figure 2).

    Revisions Figure 2.

    __Expression pattern of Lpp-Gal80>UAS-GFP in Drosophila larvae __

    Representative fluorescence microscopy images showing GFP expression driven by the Lpp-Gal80 system in Drosophila larvae. The images display dorsal (top) and ventral (bottom) views of the same larva, demonstrating the pattern of expression throughout the fat body tissue. Green fluorescence indicates cells expressing the GFP reporter under the control of the Lpp promoter, which is predominantly active in the larval fat body.

    The results on the increased number of hemocytes observed in the double NimB1, NimB4 mutant animals (Figure S3A) remains not only disconnected from the rest of the data but also unexplained. Providing a mechanistic view may require a significant amount of work that may indicate an additional role of the two NimBs but will not add to our understanding of the role of NimB1 in phagocytosis. Nevertheless, it would be at least useful to know whether in the double mutant the lymph gland is still intact, as its precocious histolysis could account for the elevated number of hemocytes. If that were the case, that could indicate that lacking the two NimBs triggers an inflammatory state that affects the lymph gland, meaning that the pathway controlling phagocytosis also has a systemic impact on development. When checking the representative Figure S4D, it seems that very large cells are present in the double mutants, even larger than in the single mutants. These could be (pre)lamellocytes, which constitute activated hemocytes, known to impact the status of the lymph gland. If the enhanced number of hemocytes does not depend on lymph gland histolysis, a simple immunolabeling with the anti-PH3 antibody would assess the proliferative phenotype of the double mutant hemocytes. At least this piece of data would provide a better explanation for the observed phenotype.

    We thank the reviewer for this interesting comment. We cannot explain why NimB1, NimB4 double mutants have more hemocytes. It is unclear to us if this is a secondary consequence of defects in efferocytosis or linked to another function of these two NimBs, such as a role in adhesion. We did look at the lymph gland and our preliminary observations suggest that NimB1, NimB4 double mutants have an easily ruptured or fragile lymph gland, which could explain the higher number and the roundish shape of hemocytes in circulation as proposed by the reviewer. Lacking expertise on lymph gland, we prefer not to include this data, as they are not central to the main message of this article on role of NimB1 on efferocytosis. We have also noted the presence of lamellocytes in unchallenged* NimB1, NimB4* double mutant larvae, as well as excessive lamellocyte production compared to controls upon clean injury (see below, Revisions Figure 3). We have mentioned the presence of lamellocytes in NimB1, NimB4 double mutants in the revised version. We prefer not include this new data directly in the article because this not central to the main message of the article.

    __Revisions Figure 3. __

    A.

    B.

    Lamellocyte recruitment following a clean injury in L3 Drosophila larvae:

    (A) Quantification of lamellocytes per 50 frames of x63 microscopy lens in *w1118 *(wild-type), NimB1crimic, NimB4sk2, NimB161, NimB4sk2, and *draperΔ5 *larvae under unchallenged conditions (UC) and 3 hours after clean injury (3h). Arrowheads indicate lamellocytes.

    (B) Representative confocal microscopy images of hemocytes isolated from challenged NimB161, NimB4sk2 larvae. Cells were fixed and stained with Phalloidin (green) to label F-actin and DAPI (blue) to visualize nuclei. The smaller inset (40x magnification) shows a detailed view of individual lamellocytes with characteristic morphology, while the larger field (20x magnification) displays the overall view on the hemocytes. Scale bar = 50 μm.

    Figure 6: The connection between the ex-vivo (Figure 5) and in vivo (Figure 6) assays should be clarified. In the first type of assay, the lack of NimB4 results in reduced internalization (while lack of NimB1 enhances it). In the in vivo assay, more fragments are seen within the cell (hence internalized), using the NimB4 mutant. Also, in the ex-vivo assay, the lack of NimB1 does not affect the first steps ('attachment' and 'membrane'), while NimB4 does, yet it is proposed that NimB1 acts in the early steps (page 11-12). In that case, wouldn't we expect the double mutant NimB1 NmB4 to have the NimB1 phenotype?

    The apparent discrepancy between our ex vivo and in vivo assays reflects the different methodologies and what each assay measures. In the ex vivo assay (Figure 4), we add exogenous fluorescently-labeled apoptotic cells to measure new engulfment events. Here, NimB4 mutant macrophages show reduced phagocytic index because they are already saturated with unresolved phagosomes, limiting their capacity to uptake additional corpses, as previously described by (Petrignani et al., 2021). This reduced uptake capacity is reflected in the decreased phagocytic index observed.

    In contrast, our in vivo assay (Figure 6) uses DAPI staining to visualize all internalized material, including previously engulfed debris. As expected, we observe accumulation of DAPI signals in NimB4 mutant macrophages under unchallenged conditions, reflecting their inability to process and clear phagosomes rather than enhanced engulfment. This phenotype highlights the role of NimB4 in phagosome maturation rather than initial uptake.

    Regarding the role of NimB1 in early phagocytic steps, while attachment and membrane measurements in the ex vivo assay don't show significant differences in NimBcrimic mutants individually, our other experiments demonstrate that NimB1 functions as a negative regulator during early recognition phases. The predominance of the NimB4 phenotype in the NimB1crimic, NimB4 double mutant parallels observations in draper mutants, where double mutants lacking both Draper I (positive regulator) and Draper II (negative regulator) display the Draper I phenotype (Logan et al., 2012). This suggests that phagosome maturation defects (the NimB4 phenotype) present a more severe bottleneck in the phagocytic process than enhanced early uptake (the NimB1crimic phenotype), explaining why the double mutant primarily exhibits accumulation of unresolved phagosomes rather than accelerated uptake. We have re-written this part of the article to clarify these points (see page 11).

    Figure 8A: a definition of the phagocytic cup mentioned in the text (page 12, 2nd paragraph) as well as the homogenization of the scale bars in Figure 8A would clarify the interpretation of Figure 8A. The structures shown for w1118 seem quite distant from the structures highlighted for NimB1crimic.

    According to reviewer 2, we have now moved this figure to the supplement. The reviewer is correct and we have modified the associated text to clarify the interpretation of the images (see page 12-13).

    The same scale should be used across different panels in Figure 8. This is particularly important since the authors mention the size of the lysotracker vesicles to conclude on their levels of maturity. This data and conclusions would be strengthened by a quantification of the vacuole sizes and the combination with markers of phagosome/lysosome maturation levels. It would help disentangling the complementary roles of NimB1 and NimB4.

    The scale bar has been homogenized.

    Minor comments:

    Figure 2BC: is there a particular reason to shift from Rp49 to Rpl32 as normalizing gene in Figure 2B and C? This prevents the comparison of NimB1 expression levels across the different graphs.

    We thank the reviewer for highlighting this point. We changed the housekeeping gene from Rp49 to RPL32 in Figure 2C to unify the normalization strategy across all experiments and allow comparisons throughout the manuscript.

    Page 9, 2nd paragraph and Figure S3C: the authors mention "Actin structure revealed an increased ratio of filopodia to lamellipodia across all mutants". A clear definition of the parameters defining filopodia and lamellipodia is required to fully appreciate the meaning of the ratio.

    We thank the reviewer for the comment. To address this comment, we have included a clear definition of the parameters used to distinguish filopodia and lamellipodia on page 9. In particular, in the revised version we now specify that filopodia were defined as thin, spike-like actin-rich protrusions, while lamellipodia were defined as broad, sheet-like structures at the cell periphery. These criteria were applied consistently for quantification.

    Figure S5B: a bar is missing in the right graph (% of cells containing AC, NimB1>UAS-NimB1-RFP). Page 10 2nd paragraph. The authors mention "draper mutants displayed impaired apoptotic cell binding and engulfment" referring to Figure 4. Figure S4 provide a more convincing illustration of this statement, since the decreased phagocytic index in Drpr KO is mostly due to less cells phagocytosing and not less material phagocytosed.

    We thank the reviewer for the careful examination. In Figure S5B, the missing bar was due to its color being too close to the background color, making it difficult to distinguish. We have now corrected this by adjusting the color to ensure it is clearly visible.

    Regarding the comment on page 10, we agree that Figure S4 more clearly illustrates the impaired apoptotic cell binding and engulfment observed in draper mutants, particularly through the reduced percentage of hemocytes engaging in phagocytosis. We have now clarified the statement in the text to ensure consistency and to guide the reader appropriately to Figure S4 (10).

    Figure 6: not easy to distinguish the DAPI labelling relative to the nucleus vs. that of apoptotic fragments.

    This is a good point. We have changed the images for clearer demonstration of the DAPI labelling. See Figure 6.

    Figure 7B: the number of cells used to generate the violin plot should be indicated in the legend or the method section.

    We have mentioned the number of cells used in the quantification (n-50 per genotype) in the figure legend.

    A schematic figure recapitulating the data would help

    We have added a schematic figure recapitulating the data. See Figure 9 with associated text.

    Page 11 last line: homeostatic rather than hemostatic.

    Thank you for this comment. We have changed it.

    Reviewer #3 (Significance (Required)):

    This study identifies a novel function for NimB1 in modulating the early stages of efferocytosis, adding to our understanding of how Nimrod proteins fine-tune apoptotic cell clearance. The authors establish a clear phenotypic contrast between NimB1 and NimB4, which provides a compelling framework for understanding how positive and negative regulators coordinate phagocytosis. It also highlights the multiple roles of the secreted members of the Nimrod scavenger receptor family, which have remained so far poorly investigated.

    This is an interesting study that could be strengthened by additional validation and broader experimental support. As the authors point out in the discussion, it is known that PS bridging molecules contribute to phagocytosis and that the contribution of positive and negative players finally tune phagocytosis in flies and mammals. Clarifying the mode of action of NimB1 in those processes would higher the impact of this interesting piece of work. For example, does NimB1 interact with NimB4 and if so, what is the role of this interaction? How does NimB1 integrate in the signaling cascade that allows scavenger receptors to bind PS? Does it act similar to Orion by enhancing the PS binding of a scavenger receptor?

    We thank the reviewer for the insightful comments and suggestions. Indeed, understanding the mode of action of NimB1 in the regulation of efferocytosis would significantly strengthen the impact of our findings. Our data, supported by structural and phylogenetic analyses, indicate that NimB1 and NimB4 share a conserved phosphatidylserine (PS)-binding motif, suggesting that these proteins may interact functionally. Preliminary biochemical observations, together with structural predictions, raise the possibility of a direct or indirect interaction between NimB1 and NimB4, although this remains to be experimentally confirmed.

    Our observations from NimB1 and NimB4 double mutants reveal that the phenotype closely resembles that of NimB4 single mutants, indicating that NimB4 plays a dominant role in the downstream maturation steps of phagosome processing. These findings are consistent with a model in which NimB1 may modulate early phagocytic uptake, possibly by competing with NimB4 for PS binding or by limiting NimB4 accessibility to apoptotic cells, thereby fine-tuning the rate of efferocytosis.

    Regarding the integration into the signaling cascade, while NimB1 and Orion both recognize PS, our data suggest that they function through distinct mechanisms. Orion enhances PS binding to Draper receptor isoforms to promote apoptotic corpse recognition. In contrast, NimB1 appears to act as an inhibitory modulator, potentially masking PS or limiting receptor engagement, thus slowing the phagocytic response. Further functional studies, including receptor-binding assays, will be important to determine whether NimB1 acts by altering receptor-ligand interactions or through a different regulatory pathway.

    Future experiments investigating the potential direct interactions between NimB1 and NimB4, their respective affinities for PS, and their influence on phagocytic receptor dynamics will be necessary to better understand NimB1’s precise mode of action. Such studies will help clarify how secreted regulators fine-tune efferocytosis in Drosophila and may offer broader insights into conserved principles of phagocytic regulation across species.

    __ __

    List of References:

    Horsthemke, M., Bachg, A. C., Groll, K., Moyzio, S., Müther, B., Hemkemeyer, S. A., Wedlich-Söldner, R., Sixt, M., Tacke, S., Bähler, M., & Hanley, P. J. (2017). Multiple roles of filopodial dynamics in particle capture and phagocytosis and phenotypes of Cdc42 and Myo10 deletion. The Journal of Biological Chemistry, 292(17), 7258–7273. https://doi.org/10.1074/jbc.M116.766923

    Ji, H., Wang, B., Shen, Y., Labib, D., Lei, J., Chen, X., Sapar, M., Boulanger, A., Dura, J.-M., & Han, C. (2023). The Drosophila chemokine–like Orion bridges phosphatidylserine and Draper in phagocytosis of neurons. Proceedings of the National Academy of Sciences, 120(24), e2303392120. https://doi.org/10.1073/pnas.2303392120

    Kurant, E., Axelrod, S., Leaman, D., & Gaul, U. (2008). Six-Microns-Under Acts Upstream of Draper in the Glial Phagocytosis of Apoptotic Neurons. Cell, 133(3), 498–509. https://doi.org/10.1016/j.cell.2008.02.052

    Lee, W. L., Mason, D., Schreiber, A. D., & Grinstein, S. (2007). Quantitative Analysis of Membrane Remodeling at the Phagocytic Cup. Molecular Biology of the Cell, 18(8), 2883–2892. https://doi.org/10.1091/mbc.E06-05-0450

    Lillico, D. M. E., Pemberton, J. G., & Stafford, J. L. (2018). Selective Regulation of Cytoskeletal Dynamics and Filopodia Formation by Teleost Leukocyte Immune-Type Receptors Differentially Contributes to Target Capture During the Phagocytic Process. Frontiers in Immunology, 9. https://doi.org/10.3389/fimmu.2018.01144

    Masters, T. A., Pontes, B., Viasnoff, V., Li, Y., & Gauthier, N. C. (2013). Plasma membrane tension orchestrates membrane trafficking, cytoskeletal remodeling, and biochemical signaling during phagocytosis. Proceedings of the National Academy of Sciences, 110(29), 11875–11880. https://doi.org/10.1073/pnas.1301766110

    Meehan, T. L., Joudi, T. F., Timmons, A. K., Taylor, J. D., Habib, C. S., Peterson, J. S., Emmanuel, S., Franc, N. C., & McCall, K. (2016). Components of the Engulfment Machinery Have Distinct Roles in Corpse Processing. PLOS ONE, 11(6), e0158217. https://doi.org/10.1371/journal.pone.0158217

    Melcarne, C., Ramond, E., Dudzic, J., Bretscher, A. J., Kurucz, É., Andó, I., & Lemaitre, B. (2019). Two Nimrod receptors, NimC1 and Eater, synergistically contribute to bacterial phagocytosis in Drosophila melanogaster. The FEBS Journal, 286(14), 2670–2691. https://doi.org/10.1111/febs.14857

    Petrignani, B., Rommelaere, S., Hakim-Mishnaevski, K., Masson, F., Ramond, E., Hilu-Dadia, R., Poidevin, M., Kondo, S., Kurant, E., & Lemaitre, B. (2021). A secreted factor NimrodB4 promotes the elimination of apoptotic corpses by phagocytes in Drosophila. EMBO Reports, 22(9), e52262. https://doi.org/10.15252/embr.202052262

    Roddie, H. G., Armitage, E. L., Coates, J. A., Johnston, S. A., & Evans, I. R. (2019). Simu-dependent clearance of dying cells regulates macrophage function and inflammation resolution. PLoS Biology, 17(5), e2006741. https://doi.org/10.1371/journal.pbio.2006741

    Serizier, S. B., & McCall, K. (2017). Scrambled Eggs: Apoptotic Cell Clearance by Non-Professional Phagocytes in the Drosophila Ovary. Frontiers in Immunology, 8, 1642. https://doi.org/10.3389/fimmu.2017.01642

    Serizier, S. B., Peterson, J. S., & McCall, K. (2022). Non-autonomous cell death induced by the Draper phagocytosis receptor requires signaling through the JNK and SRC pathways. Journal of Cell Science, 135(20), jcs250134. https://doi.org/10.1242/jcs.250134

  3. Review Commons

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    Referee #3

    Evidence, reproducibility and clarity

    This paper investigates the role of NimB1, a secreted member of the Nimrod family in Drosophila, in the process of efferocytosis, the clearance of apoptotic cells by macrophages. Previous studies have identified NimB4, another secreted Nimrod protein, as a positive regulator of efferocytosis, enhancing both apoptotic cell binding and phagosome maturation. In contrast, the authors propose that NimB1 functions as a negative regulator, slowing down the early stages of apoptotic cell binding and internalization. This regulatory balance is suggested to fine-tune efferocytosis to maintain homeostasis.

    The primary aim of this study was to characterize the function of NimB1 to better understand the roles of proteins within the NimB family.

    This study identifies a novel function for NimB1 in modulating the early stages of efferocytosis, adding to our understanding of how Nimrod proteins fine-tune apoptotic cell clearance. The authors establish a clear phenotypic contrast between NimB1 and NimB4, which provides a compelling framework for understanding how positive and negative regulators coordinate phagocytosis. It also highlights the multiple roles of the secreted members of the Nimrod scavenger receptor family, which have remained so far poorly investigated.

    This is an interesting study that could be strengthened by additional validation and broader experimental support. As the authors point out in the discussion, it is known that PS bridging molecules contribute to phagocytosis and that the contribution of positive and negative players finely tune phagocytosis in flies and mammals. Clarifying the mode of action of NimB1 in those processes would higher the impact of this interesting piece of work. For example, does NimB1 interact with NimB4 and if so, what is the role of this interaction? How does NimB1 integrate in the signaling cascade that allows scavenger receptors to bind PS? Does it act similar to Orion by enhancing the PS binding of a scavenger receptor ?

    Key Findings

    • NimB1 and NimB4 are structurally similar, as supported by AlphaFold2 modeling, suggesting functional relatedness.
    • NimB1 is expressed in macrophages, secreted into the hemolymph, and binds apoptotic cells in a phosphatidylserine (PS)-dependent manner.
    • NimB1 is induced by challenge.
    • NimB1 mutants display a hyper-phagocytic phenotype, with faster recognition and internalization of apoptotic cells.
    • NimB1 loss enhances macrophage adhesion and actin remodeling, while bacterial phagocytosis remains unaffected, suggesting a specific role in apoptotic clearance.
    • NimB1 acts early in the phagocytic process, while NimB4 functions at later stages, particularly in phagosome maturation.

    Major comments

    1. Figure 1: AlphaFold is a valuable tool for generating hypotheses, however predicted structures should not be presented as definitive evidence of similarity, particularly without complementary experimental validation. This section would be stronger if the structural predictions were explicitly framed as predictions. In the absence of such data, the interpretation should be toned down.
    2. Figure 2DE : Given its basal level in homeostatic conditions, it would have been useful to look at the NimB1-GFP upon challenge. Also, the authors show only a single larval macrophage with no comparison point. To strengthen this result, the authors could include another protein quantification method, such as western blotting. Alternatively, labelling of NimB1>UASmRFP in embryo that present the highest expression levels would also strengthen this result.
    3. In supplementary Figure S1F : the authors overexpress NimB1-RFP using the fat body driver Lpp-Gal4 and show larvae with RFP in the nephrocyte. Could filet preparations be shown? Could the authors present evidence that the Lpp driver is not expressed in the nephrocytes (or refer to literature)?
    4. The results on the increased number of hemocytes observed in the double NimB1, NimB4 mutant animals (Figure S3A) remains not only disconnected from the rest of the data but also unexplained. Providing a mechanistic view may require a significant amount of work that may indicate an additional role of the two NimBs but will not add to our understanding of the role of NimB1 in phagocytosis. Nevertheless, it would be at least useful to know whether in the double mutant the lymph gland is still intact, as its precocious histolysis could account for the elevated number of hemocytes. If that were the case, that could indicate that lacking the two NimBs triggers an inflammatory state that affects the lymph gland, meaning that the pathway controlling phagocytosis also has a systemic impact on development. When checking the representative Figure S4D, it seems that very large cells are present in the double mutants, even larger than in the single mutants. These could be (pre)lamellocytes, which constitute activated hemocytes, known to impact the status of the lymph gland. If the enhanced number of hemocytes does not depend on lymph gland histolysis, a simple immunolabeling with the anti-PH3 antibody would assess the proliferative phenotype of the double mutant hemocytes. At least this piece of data would provide a better explanation for the observed phenotype.
    5. Figure 6: The connection between the ex-vivo (Figure 5) and in vivo (Figure 6) assays should be clarified. In the first type of assay, the lack of NimB4 results in reduced internalization (while lack of NimB1 enhances it). In the in vivo assay, more fragments are seen within the cell (hence internalized), using the NimB4 mutant. Also, in the ex-vivo assay, the lack of NimB1 does not affect the first steps ('attachment' and 'membrane'), while NimB4 does, yet it is proposed that NimB1 acts in the early steps (page 11-12). In that case, wouldn't we expect the double mutant NimB1 NmB4 to have the NimB1 phenotype?
    6. Figure 8A : a definition of the phagocytic cup mentioned in the text (page 12, 2nd paragraph) as well as the homogenization of the scale bars in Figure 8A would clarify the interpretation of Figure 8A. The structures shown for w1118 seem quite distant from the structures highlighted for NimB1crimic.
    7. The same scale should be used across different panels in Figure 8. This is particularly important since the authors mention the size of the lysotracker vesicles to conclude on their levels of maturity. This data and conclusions would be strengthened by a quantification of the vacuole sizes and the combination with markers of phagosome/lysosome maturation levels. It would help disentangling the complementary roles of NimB1 and NimB4.

    Minor comments:

    Figure 2BC : is there a particular reason to shift from Rp49 to Rpl32 as normalizing gene in Figure 2B and C? This prevents the comparison of NimB1 expression levels across the different graphs. Page 9, 2nd paragraph and Figure S3C: the authors mention "Actin structure revealed an increased ratio of filopodia to lamellipodia across all mutants". A clear definition of the parameters defining filopodia and lamellipodia is required to fully appreciate the meaning of the ratio. Figure S5B: a bar is missing in the right graph (% of cells containing AC, NimB1>UAS-NimB1-RFP). Page 10 2nd paragraph. The authors mention "draper mutants displayed impaired apoptotic cell binding and engulfment" referring to Figure 4. Figure S4 provide a more convincing illustration of this statement, since the decreased phagocytic index in Drpr KO is mostly due to less cells phagocytosing and not less material phagocytosed. Figure 6: not easy to distinguish the DAPI labelling relative to the nucleus vs. that of apoptotic fragments. Figure 7B: the number of cells used to generate the violin plot should be indicated in the legend or the method section. A schematic figure recapitulating the data would help. Page 11 last line: homeostatic rather than hemostatic.

    Significance

    This study identifies a novel function for NimB1 in modulating the early stages of efferocytosis, adding to our understanding of how Nimrod proteins fine-tune apoptotic cell clearance. The authors establish a clear phenotypic contrast between NimB1 and NimB4, which provides a compelling framework for understanding how positive and negative regulators coordinate phagocytosis. It also highlights the multiple roles of the secreted members of the Nimrod scavenger receptor family, which have remained so far poorly investigated.

    This is an interesting study that could be strengthened by additional validation and broader experimental support. As the authors point out in the discussion, it is known that PS bridging molecules contribute to phagocytosis and that the contribution of positive and negative players finely tune phagocytosis in flies and mammals. Clarifying the mode of action of NimB1 in those processes would higher the impact of this interesting piece of work. For example, does NimB1 interact with NimB4 and if so, what is the role of this interaction? How does NimB1 integrate in the signaling cascade that allows scavenger receptors to bind PS? Does it act similar to Orion by enhancing the PS binding of a scavenger receptor ?

  4. Review Commons

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    Referee #2

    Evidence, reproducibility and clarity

    Summary:

    In this study, Dolgikh and colleagues propose a first investigation about the role of the drosophila Nimrod protein NimB1. Although the role of several members of the family in phagocytosis has been explored, the function of Nimrod type B proteins is less addressed. With in silico analysis, they first see a strong similarity between NimB1 and NimB4. They show that NimB1 is primarily expressed in phagocytes, and as NimB4 can bind phosphatidylserines (PS), leading to a possible shared role in efferocytosis. Using transgenic and null drosophila models, the authors then compare the impact of NimB1 overexpression or deficiency. They compare the effects shown to NimB4 and Draper deficient lines, as these two proteins were previously shown to play a role in efferocytosis. They propose that NimB1 is a secreted protein that binds apoptotic cells. They show that NimB1 deficiency changes the adhesion properties of macrophages. The major finding is that NimB1 delays the early stages of efferocytosis, contrary to NimB4 and Draper that on the contrary facilitate efferocytosis. In contrast, the authors propose that NimB1 increases the formation of phagosomes.

    Major comments:

    • One of the major technical challenges here was to generate models to allow the detection of the protein in cellulo and in vivo. Although the results are convincing in transgenic lines NimB1 expression is driven by the endogenous promoter, one could still argue that the GFP tags would lead to changes in the localization of the protein.
    • In line with the previous comment, to show that NimB1 is a secreted protein, the authors use an overexpression model. How to be sure, that overexpression itself does not lead to increased secretion, or shedding from the membrane ?
    • Would an experiment with a control consisting in a known protein secreted by macrophages lead to the same staining pattern (positive control)? Could another methodology like a Western Blot on supernatants from hemocyte cell culture (over)expressing NimB1, with an anti-RFP staining, be envisaged?
    • It sems counterintuitive that phagocytes from Draper and NimB4 null mutants with defects in efferocytosis show increased load of apoptotic cells (Figure 6C and D in both unchallenged and injury condition). Do the authors have precedent data to cite going to the same direction? Are cell debris engulfed but not degraded efficiently?
    • In Figure 6D it seems indeed that NimB4, NimB1/NimB4 and Draper mutants do not accumulate more apoptotic material upon injury. However, levels for NimB4 is close to the one obtained with NimB1 mutants. Is it statistically true? If yes, what could be the reason for this similarity ? In any case, as some important conclusion relies on the comparison between UC and injury conditions, adequate statistics and representations could be proposed.
    • The authors claim with analyses of Figure 8C and D, that NimB1 mutants show acidic vehicles normal in size and fluorescence intensity. However, statistical differences are still observed compared to control condition, which is also seen in representative images shown.

    Minor comments:

    • In figure 2D, what allows to say the expression is restricted in macrophages ? Is it the colocalization with SIMU being a macrophage-specific marker?
    • In figure S3B and C, it appears that double NimB1/NimB4 mutants exhibit less spreading than single ones (especially NimB4). Is it the case (statistic significance). If yes what could be the explanation?
    • Several graphs are identical between figure 4 ans S4. It is probably not useful and complicates reading.
    • As TEM images shown in Figure 8B do not lead to quantitative data, I would put it as supplementary file.

    Significance

    This study uses several approaches and models to address the role of NimB1 in efferocytosis. Both In Vitro and In Vivo approaches are proposed. They give insight into the role of this protein with unknown function so far. Some statistical analysis could be performed to improve the clarity of conclusions. One of the important aspects is the secreted nature of NimB1.However, additional approaches could be proposed to confirm this.

    Basic immunologists and cell biologists would be interested in reading this article that highlights the delicate equilibrium between pro and anti-efferocytosis molecules.

    I am an immunologist/cell biologist with expertise in lysosomal catabolism. As I work on mouse models or Human samples, my mastering of drosophila as a model is limited.

  5. Review Commons

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    Referee #1

    Evidence, reproducibility and clarity

    This work defines NimB1 protein as a PS binding bridging molecule but with a negative regulatory role in efferocytosis. Specifically, the authors demonstrate via a variety of genetic, cell biological, and other approaches that loss of NimB1 leads to Drosophila macrophages being more adherent to apoptotic targets and engulf them more robustly. The authors also nicely demonstrate that the function of NimB1 differs from NimB4, and the double mutant demonstrating PS-binding yet, distinct roles. Further, the authors show that NimB1 does not affect bacterial phagocytosis.

    Overall, this is a well-done study. The authors have already done a very thorough job addressing the key points and I congratulate the authors.

    My only minor comment is that the authors could try to make the comment better about whether or not such a 'negative regulatory' bridging molecules may exist in other species, and particularly mammals. If so, this is quite novel. The authors refer to CD47 but this is a membrane protein. The other minor comment is whether the authors ever tried express the PS binding domains as a fusion protein - this would provide a more direct evidence for the binding to PS (although the authors do competitive inhibition with Annexin V). This could be commented upon although testing this is not necessary if they have not already done so.

    Significance

    The identification of the negative regulator bridging protein NimB1 is novel and could be broadly interesting to those studying efferocytosis.

  6. Version published to 10.1101/2025.01.07.631694 on bioRxiv