Triggering MSR1 promotes JNK‐mediated inflammation in IL‐4‐activated macrophages

Abstract Alternatively activated M2 macrophages play an important role in maintenance of tissue homeostasis by scavenging dead cells, cell debris and lipoprotein aggregates via phagocytosis. Using proteomics, we investigated how alternative activation, driven by IL‐4, modulated the phagosomal proteome to control macrophage function. Our data indicate that alternative activation enhances homeostatic functions such as proteolysis, lipolysis and nutrient transport. Intriguingly, we identified the enhanced recruitment of the TAK1/MKK7/JNK signalling complex to phagosomes of IL‐4‐activated macrophages. The recruitment of this signalling complex was mediated through K63 polyubiquitylation of the macrophage scavenger receptor 1 (MSR1). Triggering of MSR1 in IL‐4‐activated macrophages leads to enhanced JNK activation, thereby promoting a phenotypic switch from an anti‐inflammatory to a pro‐inflammatory state, which was abolished upon MSR1 deletion or JNK inhibition. Moreover, MSR1 K63 polyubiquitylation correlated with the activation of JNK signalling in ovarian cancer tissue from human patients, suggesting that it may be relevant for macrophage phenotypic shift in vivo. Altogether, we identified that MSR1 signals through JNK via K63 polyubiquitylation and provides evidence for the receptor's involvement in macrophage polarization.


Summary
Alternatively activated M2 macrophages (AAMs) play an important role in maintenance of tissue homeostasis by scavenging dead cells and cell debris via phagocytosis. An essential step in this process is phagosomal maturation. Using high-45 resolution LC-MS/MS, we investigated how alternative activation, driven by IL-4, modulated the phagosomal proteome to control macrophage function. One of the most intriguing insights was the recruitment of the TAK1/MKK7/JNK signalling complex to the phagosomes of IL-4 activated macrophages. The recruitment of this signalling complex was mediated through K63 polyubiquitylation of the macrophage 50 scavenger receptor 1 (MSR1). MSR1 activation induced JNK signalling, thereby facilitating macrophage polarization towards an M1 pro-inflammatory state, which was abolished upon MSR1 deletion or JNK inhibition. Moreover, MSR1 K63 polyubiquitylation correlated with the activation of JNK signalling in ovarian cancer tissue from human patients, suggesting that it may be relevant for macrophage 55

Introduction
Phagocytosis is a highly conserved process essential for host defence and tissue remodelling. It involves the recognition of particles by a variety of cell surface receptors, followed by cargo processing and delivery to lysosomes via phagosome-65 lysosome fusion, process known as phagosome maturation. This leads to gradual acidification of the phagosomal lumen and acquisition of digestive enzymes required for the degradation of phagosomal cargo. Therefore, phagocytosis is not only responsible for elimination of bacterial pathogens, but also the clearance of apoptotic cells, cell debris and senescence cells and orchestrates the subsequent immune 70 response (Lemke, 2013, Lemke, 2017, Murray & Wynn, 2011, Rothlin et al., 2007.
Central to this process is phagosomal maturation. If uncontrolled, the inappropriate clearance of apoptotic bodies can give rise to autoimmune diseases, atherosclerosis and cancer, while failure to ingest or kill pathogens can result in deadly infections (Arandjelovic & Ravichandran, 2015, Colegio et al., 2014, Johnson & Newby, 2009, 75 Nagata et al., 2010. Therefore, it is of great importance to understand which signalling pathways regulate phagocytosis and phagosomal maturation.
It has recently been acknowledged that the phagosome serves as a signalling platform and interacts with innate immune signalling (Heckmann et al., 2017, Kagan, 2012, Martinez et al., 2011, Martinez et al., 2015, Stuart et al., 2007. However, 80 whether phagosome-associated cell signalling is independent of its role in cargo degradation has not been well understood. Supporting this notion, recent proteomic studies demonstrated that phagosomes are dynamic organelles that change their composition and function in response to infection or inflammation (Boulais et al., 2010, Hartlova et al., 2018, Naujoks et al., 2016, Trost 85 et al., 2009. While the regulation of phagosomal maturation in so-called M1 inflammatory macrophages has been extensively studied, the mechanisms facilitating phagosomal maturation in macrophages involved in tissue repair remains poorly understood (Balce DR., Keizer. S.J., 2011) Th2-derived cytokines, such as interleukin-4 (IL-4) and interleukin-13 (IL-13) 90 induce a strong anti-inflammatory macrophage phenotype, also called alternative activated macrophages (M2). M2 macrophages and tissue resident macrophages, which often resemble an M2-like state, clear cell debris and dead cells through phagocytosis. They are therefore essential for maintenance and tissue homeostasis.

M2 alternatively activated macrophages (AAMs) inhibit inflammatory responses and 95
promote angiogenesis and tissue repair by synthetizing mediators required for collagen deposition, which is important for wound healing (Gordon & Martinez, 2010).
It has been shown that IL-4 enhanced phagosomal protein degradation (Balce et al., 2011). Whether IL-4 regulates other phagosomal functions, and through which molecular mechanisms, remains unclear. 100 Here we demonstrate that the TAK1/MKK7/JNK signalling complex showed an enhanced association with the phagosome upon IL-4 macrophage activation using an unbiased quantitative LC-MS/MS approach. We further show that the assembly of the signalling complex is mediated through K63 polyubiquitylation. By combining K63polyubiquitylation enrichment and mass spectrometry approaches, we identified 105 macrophage scavenger receptor 1 (MSR1) as the upstream receptor that promotes the recruitment of the TAK1/MKK7/JNK signalling complex to the phagosome.
Triggering MSR1 induces JNK activation in M2 macrophages. This MSR1/JNK signalling pathway activation leads to a M2/M1 macrophage phenotypic switch that is abolished in macrophages lacking MSR1. We demonstrate that MSR1 is K63 ubiquitylated and 110 signals through JNK in human patient ovarian cancer, thus suggesting a potential role in human cancer.

115
Alternative activation regulates phagosomal proteolysis and lipol ysis To determine the impact of IL-4 on phagocytosis and phagosomal functions, we examined the rate of phagocytosis and phagosomal maturation in IL-4 AAMs (M2) and resting macrophages (M0). We found that M2 macrophages have enhanced uptake of 120 apoptotic cells, while uptake of necrotic cells was comparable to M0 resting macrophages ( Figure 1A). To determine whether the enhanced uptake was because of the negative charge of apoptotic cells, we compared the uptake of fluorescentlylabelled carboxylated negatively charged and amino positively charged silica microsphere in M2 and M0 macrophages. The analysis revealed an increased uptake 125 of negatively charged microspheres in M2 macrophages, while the engulfment of positive charged microsphere was similar to M0 macrophages indicating a similarity of carboxylated microspheres with apoptotic cells (Figure 1B). Next we analysed the functional parameters of the phagosomal lumen including proteolysis, acidification and lipolysis using real-time fluorescence assays (Podinovskaia et al., 2013, Yates et 130 al., 2005. Consistent with the previous reports, we observed enhanced proteolytic activity in phagosomes of M2 macrophages (Balce et al., 2011). Furthermore, we found that IL-4 increased phagosomal lipid degradation and facilitated phagosomal acidification ( Figure 1C-E) indicating that IL-4 activation promotes ability of macrophages to degrade lipid-rich particles through phagosomes. 135

Quantitative proteomics analysis of phagosomes from IL-4 alternatively activated macrophages
To obtain further molecular details about the changes on phagosomes of M2 macrophages, we isolated highly pure phagosomes from M2 and M0 macrophages by 140 floatation on a sucrose gradient using carboxylated microspheres and analysed their proteomes by quantitative LC-MS/MS (Figure 1A, S2A) (Desjardins et al., 1994, Trost et al., 2009. Comparative analysis led to the identification of 20,614 distinct peptides corresponding to 1,948 unique proteins across three independent replicates at a falsediscovery rate (FDR) of <1%, of which 1,766 proteins were quantified in at least two of 145 the three biological replicates. IL-4 activation induced strong changes to the phagosome proteome with 121 proteins significantly up-and 62 proteins significantly down-regulated (2-fold change, p<0.05) ( Figure 2B; Table S1), some of which we validated by Western blot analysis ( Figure S2B). Consistent with the above observations, a subset of proteins involved in lipid metabolism (Lpl lipoprotein lipase, 150 ABHD12 lipase and phospholipase D1), acidification (v-ATPase complex) and lysosomal enzymes including cathepsins L1 and D were highly enriched on the phagosome of M2 macrophages ( Figure 2D, Table S1). Moreover, GO-term ( Figure 2E) and protein network analysis ( Figure S2C) further showed that IL-4 alternative activation also increased phagosome abundance of scavenger receptors such as MARCO, CD36, 155 Colec12 and MSR1 required for clearance of dead cells while Toll-like receptors (TLRs) involved in inflammatory response were reduced (Figure 2E, S2C). Consistent with previous report, superoxide anion generation including the NADPH oxidase complex proteins NCF1 (p47-phox), Cyba (p22-phox), Cybb (gp91-phox) and superoxide dismutase SOD1 were strongly down-regulated in phagosomes from M2 macrophages 160 ( Figure 2E, Figure S2C) (Balce et al., 2011). Interestingly, M2 macrophages also enriched a large number of specific carbohydrate-binding proteins such as lectins, while carbohydrate hydrolases were significantly reduced. This indicates a conservation of phagocytosed glycans, potentially for antigen presentation via MHC class II molecules (Avci et al., 2013). Furthermore, M2 phagosomes showed higher 165 phosphatidylinositol-binding proteins, suggesting changes to the phagosome membrane lipid content. Taken together, these results indicate that the phagosome of M2 macrophages has mainly a homeostatic role with its increased ability to hydrolyse proteins and lipids of incoming cargo.

TAK1/MKK7/JNK are recruited to the phagosome of M2 macrophages via K63 polyubiquitylation
Interestingly, anti-inflammatory IL-4 activation also led to an increased phagosome abundance of the pro-inflammatory MAP-kinase signalling complex around TAK1 175 (Map3k7, 2.1-fold) and MKK7 (Map2k7, 3.1-fold) (Table S1, Figure S2C) indicating cross-regulation between anti-and pro-inflammatory pathways. Given the increased abundance of these pro-inflammatory kinases was surprising on phagosomes of IL-4stimulated macrophages, we next investigated how this complex was translocated to the phagosome. Immunoblot analyses of total cell lysates and phagosomal fractions 180 revealed significant enrichment of TAK1 and MKK7 on phagosomes of M2 macrophages compared to resting M0 macrophages (Figure 3A, B). Activated TAK1 can phosphorylate two MAPK kinases, MKK4 and MKK7, both implicated in the activation of JNK. While MKK4 can activate p38 and JNK MAPKs signalling pathways, MKK7 selectively activates JNK (Tournier et al., 2001). Noteworthy, our mass 185 spectrometry data revealed that only MKK7 was enriched on phagosomes upon IL-4 alternative activation. Consistent with our LC-MS/MS data, MKK4 was not detected on phagosomes of M2 macrophages by immunoblot analysis indicating that MKK7 alone was important in this phagosome signalling pathway. Further immunoblot analysis also confirmed enrichment of JNK of M2 macrophage phagosomes. 190 Previous data have shown that upon pro-inflammatory Interleukin-1 receptor or Toll-like receptor (TLR) activation, the TAK1/MKK7/JNK complex binds to the TAB1/TAB2 protein complex, which in turn is recruited to K63 polyubiquitin chains (Emmerich et al., 2013, Xia et al., 2009. We next tested whether TAB1/TAB2 are As it is well-established that TAK1 binds via TAB1/2/3 to free and proteinanchored K63-polyubiquitin chains in inflammatory innate immune responses (Emmerich et al., 2013, Xia et al., 2009, we tested whether phagosomes from M2 macrophages contain K63-polyubiquitylated proteins independent of inflammatory stimuli. Immunoblot analysis of phagosome extracts probed with anti-K63 205 polyubiquitin antibodies revealed that phagosomes contain a large amount of K63polyubiquitylated proteins compared to the total cell lysate, which was even more increased by alternative activation (Figure 3C). To determine whether recruitment of TAB1, TAB2, TAK1 and MKK7 to the M2 phagosome was indeed K63 polyubiquitylation-dependent, we treated cells with NSC697923, a pharmacological 210 inhibitor of the K63-specific E2 conjugating enzyme UBC13-UEV1A (Pulvino et al., 2012) and probed isolated phagosomes for K63 polyubiquitylation. As shown in Figure   3D, recruitment of the protein complex was virtually abolished under these conditions. These data indicate that IL-4 activation of macrophages promotes K63 polyubiquitylation which recruits the TAK1/MKK7/JNK complex to the phagosome. 215

Macrophage scavenger receptor 1 is K63 polyubiquitylated and interacts with TAK1/MKK7/JNK on the phagosome of M2 macrophages
To identify the K63 polyubiquitylated associated with TAK1/MKK7/JNK 220 complex on the phagosome of M2 macrophages, we enriched polyubiquitylated phagosomal proteins from M2 macrophages using tandem ubiquitin-binding entities (TUBEs) of a repeat of the Npl4 Zinc Finger (NZF) domain of TAB2 tagged with Halo (termed here Halo-TAB2) that bind to K63 polyubiquitin chains ( Figure 4A) (Emmerich et al., 2013, Heap et al., 2017, Hjerpe et al., 2009. Quantitative mass spectrometric 225 analysis of these pull-downs identified 538 proteins that were reproducibly captured by Halo-TAB2 compared to mutant Halo [T674A/F675A] TAB2 control beads (based on a 2-fold, p<0.05 cut-off) ( Table S2). Moreover, we identified 62 novel direct ubiquitylation (-GlyGly) sites on 33 different proteins. Quantitation of the data revealed that the Gly-Gly peptide derived from K63-linked polyubiquitin was by far the 230 most abundant, proving that we achieved good enrichment. However, we also  Table S3). 240 Interestingly, one of the most abundant Gly-Gly-modified peptides was a peptide containing lysine 27 (K27) of macrophage scavenger receptor 1/scavenger receptor A (MSR1/SR-A; CD204). This site is highly conserved between human and mouse ( Figure 4C). MSR1 is a multifunctional phagocytic receptor, highly expressed in macrophages, involved in uptake of apoptotic cells and modified lipoproteins (Kelley 245 et al., 2014). In addition to its scavenging function, MSR1 has been implicated in the innate immune response to bacteria (Platt & Gordon, 2001).
Our MS and immunoblot data showed an increase of MSR1 on phagosomes from M2 macrophages compared to M0 macrophages (Table S1, Figure S4A), while both total cell and cell surface expression levels of MSR1 were unchanged between 250 the two conditions ( Figure S4B). However, when we precipitated K63polyubiquitylated proteins from resting and M2 macrophages, we could see that the polyubiquitylated forms of MSR1 were considerably more abundant in alternatively activated macrophages (Figure 4D), suggesting that MSR1 becomes polyubiquitylated in M2 macrophages. 255 To validate K63 polyubiquitylation of MSR1, we treated enriched polyubiquitylated phagosome protein extracts from M2 macrophages with the K63specific deubiquitylase (DUB) AMSH-LP or the non-specific DUB USP2 (Ritorto et al., 2014) ( Figure 4E). In both cases, the high molecular smear of ubiquitylated MSR1 decreased while the band representing the non-ubiquitylated form of MSR1 increased 260 significantly, indicating that MSR1 was predominantly K63 polyubiquitylated on phagosomes upon uptake of carboxylated beads in M2 macrophages.
We next investigated whether K63-polyubiquitylated MSR1 might recruit the TAB1/TAB2/TAK1/MKK7 complex to the phagosome. To test this, we pulled down MSR1 from extracts of carboxylated bead phagosomes. We found that indeed 265 Figure 4F).
Moreover, using a different antibody against TAK1, immunoblot analysis patterns indicated also substantial post-translational modification -most likely polyubiquitylation -of TAK1, which was considerably enhanced in MSR1 IPs. It has been reported that ubiquitylation of TAK1 activated the kinase activity (Fan et al., 270 2010), suggesting that TAK1 and the downstream kinase MKK7 are recruited in the active state or activation is triggered by binding to K63 polyubiquitylated MSR1. Taken together, these data demonstrate that MSR1 becomes K63 polyubiquitylated upon activation in IL-4-activated macrophages which recruits the TAK1/MKK7 kinase signalling complex. 275

Triggering MSR1 activates JNK pathway
To further test whether the engagement of MSR1 causes the activation of the TAK1/MKK7/JNK pathway, wild-type (WT) and MSR1 KO M2 macrophages were 280 stimulated with the MSR1 ligands fucoidan and oxidized LDL (oxLDL) and analysed for MSR1 K63-polyubiquitylation and activation of JNK signalling (Greaves & Gordon, 2009). The analysis revealed that M2 macrophages deficient in MSR1 showed signalling pathway, which induces a pro-inflammatory phenotype switch in these macrophages.

MSR1 is polyubiquitylated in human tumour associated macrophages
Next, we wanted to test if MSR1 ubiquitylation was also present in settings of human disease. MSR1 has been implicated in tumour development and progression 305 (Chanmee et al., 2014, Komohara et al., 2009) and tumour-associated macrophages (TAMs) have been shown to resemble M2 macrophage phenotype with MSR1 protein expression (Sica et al., 2007). However, the role of MSR1 in TAMs is not fully understood. We characterised five human patient samples with different types of cancers for the presence of MSR1. These tissue samples showed a high variability in 310 the number of TAMs that stained positively for MSR1 using immunohistochemistry ( Figure 6A). Consistent with the human proteome atlas data (http://www.proteinatlas.org/ENSG00000038945-MSR1/cancer), we found particularly high expression of MSR1 in patient ovarian cancer. Interestingly, the TAMs of the patient with ovarian cancer also showed increased levels of ubiquitylated MSR1 315 as well as enhanced phosphorylation of JNK and its substrate c-Jun ( Figure 6B). This suggests that JNK signalling downstream of poly-ubiquitylated MSR1 is present in TAMs of human cancers and could potentially be involved in tumour promotion.

320
Macrophages are highly diverse and plastic immune cells that can polarise in response to environmental cues into many different phenotypes. Because of their important and diverse functions in regulating immune responses and metabolism, dysregulated macrophage polarization is frequently associated with disease (Schultze et al., 2015). Here we expand our understanding of macrophage phenotype switching 325 by showing on the molecular level that engagement of MSR1 in IL-4-activated macrophages results in the activation of the JNK signalling pathway, thereby inducing a shift from an anti-inflammatory to a proinflammatory phenotype.
Recent data implies that distinct pathways regulate uptake kinetics of different particles as well as phagosome functions in macrophages and these are further 330 controlled by macrophage activation. It was demonstrated that both the phagocytic receptors (Balce et al., 2016 and pro-inflammatory (Ghigo et al., 2010, Trost et al., 2009, Yates et al., 2007 and anti-inflammatory activation (Balce et al., 2011, Varin et al., 2010 of macrophages affects phagosome functions and these are regulated by signalling pathways such as kinases (Hartlova et al., 2018) and unpublished data).
A striking finding from our study is the identification of increased ubiquitylation on the phagosome. Ubiquitylation in the endo-lysosomal system is generally thought to be important for lysosomal degradation of membrane proteins (Piper et al., 2014). Our data indicates that K63-polyubiquitylation on phagosomal 340 proteins is also used as a scaffold for the recruitment of signalling complexes, in particular the kinase complex TAK1/MKK7/JNK to MSR1 via specific polyubiquitylation on a conserved lysine K27. Remarkably, we could show that only MKK7 and not MKK4 is recruited to this signalling complex in IL-4-activated macrophages, showing a different role for the two MAP kinase kinases in activating JNK. 345 It is noteworthy that MSR1 is in almost equal abundance in resting, alternatively and classically activated macrophages. This indicates that macrophage activation (as signal 1) induces activation or transcription or translocation of an E3 ligase. This unknown E3 ligase then ubiquitylates, after MSR1 ligation (signal 2), the receptor, enabling proinflammatory signalling through the TAK1/MKK7/JNK signalling 350 complex ( Figure 6C). During inflammation, this signalling probably does not add substantially to the macrophage phenotype as TLR or IL1R activation induces a much stronger pro-inflammatory signal. However, in M2 or tissue-resident macrophages with a similar M2-like phenotype, prolonged MSR1 ligation could lead to a proinflammatory switch of these macrophage subsets via JNK. Interestingly, recent data 355 in Drosophila showed that apoptotic corpses prime macrophages for detection of tissue damage and that this priming and subsequent recruitment to wounds was dependent on JNK (Weavers et al., 2016). This suggests that JNK activation induced by uptake of apoptotic bodies could regulate various responses.
MSR1 has in recent years been established as a good marker for TAMs 360 (Allavena & Mantovani, 2012) which resemble rather a M2 alternatively activated phenotype and have been associated with tumour promotion (Sica et al., 2007). While previously it was shown that lack of MSR1 delayed the growth of EL4 lymphoma in mice by increased pro-inflammatory responses to necrotic cells (Komohara et al., 2009), our data revealed an increased expression of K63-polyubiquitylated MSR1 in a 365 human ovarian cancer. This coincided with increased activation of JNK proinflammatory signalling pathway, suggesting that the MSR1-JNK signalling pathway is activated in the progression of cancer. Interestingly, MSR1 has been previously shown to promote tumour progression and metastasis in ovarian and pancreatic cancer mouse models (Neyen et al., 2013), suggesting that MSR1 and downstream signalling 370 may be a potential drug target in the prevention of cancer metastasis progression.

Antibodies 380
The following antibodies were purchased from Cell Signalling Technology: pSTAT1

Culturing and activation of bone marrow-derived macrophages
Bone marrow cells were collected from femurs and tibiae of 6-8 week old C57BL/6 wild type (WT) or MSR1/SR-A knock-out mice (kindly provided by Siamon Gordon). 400 The cells were treated with red blood cell lysis buffer (155 mM NH4Cl, 12 mM NaHCO3, 0.1 mM EDTA) and plated on tissue culture plastic (Corning Incorporated) for 3 days in DMEM (Gibco) containing 10% FBS, 2 mM glutamine, 100 units/ml Penicillin-Streptomycin (Gibco), and 20% L929 conditioned supplement. At day three, the cells in supernatant were transferred to untreated 10 cm Petri dishes (BD Biosciences) for 405 seven days for the differentiation into bone marrow-derived macrophages (BMDMs).
Cells were then harvested on ice, washed in cold PBS, and phagosomes were isolated 415 as described in previous works , Trost et al., 2009. Enriched protein extracts contained less than 5% contamination from other cellular organelles as estimated from immunoblotting experiments ( Figure S2A).
For pull down assays, phagosomes were isolated using 1 µm magnetic beads (Estapor/Merck). Magnetic beads were diluted 1:300 in complete DMEM media and 420 incubated with BMDMs for 30 min.

Phagosome functional assays
Fluorogenic assays for phagosomal proteolysis, acidification and lipolysis were adapted from the method from the Russell laboratory (Podinovskaia et al., 2013, VanderVen et al., 2010, Yates et al., 2005. For proteolysis and acidification BMDMs were plated onto 96-well plates at 1 × 10 5 cells per ml 24 h prior to the experiment.

Proteome quantification and bioinformatics analysis
The phagosome proteomics dataset of IL-4-activated macrophages was extracted A reverse database was used for false peptide discovery. Mass accuracy was set to 10 500 ppm for precursor ions and 0.5 Da for ion trap MS/MS data. Identifications were filtered at a 1% false-discovery rate (FDR) at the protein and peptide level, accepting a minimum peptide length of 7. Quantification used only razor and unique peptides, and required a minimum ratio count of 2. "Re-quantify" and "match between runs" were enabled. Normalized ratios were extracted for each protein/condition and used 505 for downstream analyses.
Statistical analyses were performed in Perseus (v1.3.0.4). T-test-based statistics were applied on normalized and logarithmized protein ratios to extract the significant regulated proteins. Hierarchical clustering was performed in Perseus on logarithmized ratios of significant proteins using Correlation distances and Average linkage to 510 generate the heatmap.

GO term and Network analyses
The H/L log fold changes for all quantifiable proteins in each condition (in replicates) were tested against the null hypothesis that the mean log fold change was zero. We 515 used a one-sample t-test with shrinkage variance of Opgen-Rhein & Strimmer (Opgen-

Rhein & Strimmer, 2007). Each protein was annotated with GO-terms from Mouse
Genome Informatics Database (downloaded on 5/11/2014). Log fold change of each GO-term was calculated as the mean of log fold changes of all proteins annotated with this GO-term. The significance of this mean, against the null hypothesis that the mean 520 is zero (i.e., there is no discernible fold change in the GO-term proteins), was found using a bootstrap technique. A protein sample of the same size as the GO-term group was randomly selected (without replacement) from the pool of all quantifiable proteins and its mean log fold change found. The sampling process was repeated 100,000 times and the significance p-value was determined as the percentile of 525 bootstraps where the absolute log fold change was greater than in the GO-term group.
Proteins annotated with given GO-terms are presented in 'volcano plots', showing, for each protein, the mean log fold change of replicates versus the p-value. The error bars represent the shrinkage standard error.
After incubation with HRP-labeled secondary antibodies, proteins were detected using ECL and X-ray films. Immunoblots were quantified in ImageJ software.

Immunofluorescence
Resting and alternative-activated BMDMs were seeded at 1 × 10 5 /ml on glass coverslips. Silica beads (3 μm, Kisker Biotech) were phagocytosed for 30 min by using a dilution of 1:1000 in cell culture media. Cells were subsequently fixed in 4% paraformaldehyde (Affymetrix) and permeabilized by incubating for 5 min with PBS 545 containing 0.02% NP-40. Rabbit anti-Rab7a antibody was used at dilution of 1:300 to indicate the phagosomes. To visualize the phagosomal location of interested proteins, sheep anti-TAK1/TAB1/TAB2/MKK7 antibodies (DSTT) were used at 4 μg/ml. Cells were imaged in a Zeiss LSM 700 confocal microscope using a x100 Plan Apochromat objective (NA 1.46) and an optical section thickness of 0.7 µm. For quantitation, all 550 laser, pin-hole and gain, etc. settings kept the same for all images. Fields of cells were selected at random using only the DAPI stained channel. Optical sections were taken through the centre of the cell including the beads and 10 fields collected per coverslip.
A region of interest (ROI) was drawn around each bead-containing phagosome, the Rab7a ring associated with the bead was included. The green intensity was collected 555 for the same ROI. The integrated sum of the red and green intensities in each ROI were collected and expressed as a ratio. At least 25 individual phagosomes were analysed for each protein target. DAPI, Green-Alexa 488 (target antigen), Red-Alexa 594 (Rab7a) and DIC channels were all collected and images were quantified using the Volocity programme (Perkin-Elmer). 560

Deubiquitylation assay 580
The polyubiquitylated proteins captured by Halo-TAB2 beads were washed twice in reaction buffer (50 mM Tris pH 7.5, 50 mM NaCl, 2 mM DTT). The beads were then incubated with or without AMSH-LP (5 μM) or USP2 (1 μM) in 30 μl reaction buffer at 30 °C for 1 h. The reaction was quenched by denaturation in 1% LDS. Eluted proteins were separated on SDS-PAGE and immunoblotted with anti-MSR1 or anti-K63 pUb 585 chain antibodies.

MSR1 co-immunoprecipitation from phagosome extracts
Rabbit anti-MSR1 antibody and rabbit IgG were coupled to protein A-Sepharose

Immunohistochemistry 635
Three micrometer tissue sections from selected paraffin blocks of primary human tumours were prepared. Slides were incubated overnight at 56°C, deparaffinized in xylene for 20 min, rehydrated through a graded ethanol series, and washed with PBS.
Immunohistochemistry was performed on a Ventana Benchmark XT automatic immunostaining device (Roche). A heat induced epitope retrieval step was performed in Ventana CC1 solution for 60 minutes. Primary antibodies were incubated for 40 minutes, 60 minutes and 120 minutes, respectively. An Ultravision detection system was used.

Statistical analysis
Statistical analysis was performed using GraphPad Prism software. Definition of statistical analysis and post hoc tests used can be found in figure legends. The statistical significance of data is denoted on graphs by asterisks (*) where *P < 0.05, **P < 0.01, ***P < 0.001 or ns = not significant.

Acknowledgements:
We would like to thank the DNA cloning, Protein Production, Antibody     Working model: MSR1 is activated by ligation through many different substrates, including apoptotic cells, fucoidan or oxidised LDL (Signal 1). However, only when the macrophage is IL-4-activated (Signal 2) becomes MSR1 ubiquitylated by an unknown E2/E3 ligase. This ubiquitylation recruits Tab2/3, Tak1, Mkk7 and finally JNK, thereby 940 allowing MSR1 to signal directly through the JNK signalling pathway which induces pro-inflammatory gene transcription.