TPL‐2 kinase induces phagosome acidification to promote macrophage killing of bacteria

Abstract Tumour progression locus 2 (TPL‐2) kinase mediates Toll‐like receptor (TLR) activation of ERK1/2 and p38α MAP kinases in myeloid cells to modulate expression of key cytokines in innate immunity. This study identified a novel MAP kinase‐independent regulatory function for TPL‐2 in phagosome maturation, an essential process for killing of phagocytosed microbes. TPL‐2 catalytic activity was demonstrated to induce phagosome acidification and proteolysis in primary mouse and human macrophages following uptake of latex beads. Quantitative proteomics revealed that blocking TPL‐2 catalytic activity significantly altered the protein composition of phagosomes, particularly reducing the abundance of V‐ATPase proton pump subunits. Furthermore, TPL‐2 stimulated the phosphorylation of DMXL1, a regulator of V‐ATPases, to induce V‐ATPase assembly and phagosome acidification. Consistent with these results, TPL‐2 catalytic activity was required for phagosome acidification and the efficient killing of Staphylococcus aureus and Citrobacter rodentium following phagocytic uptake by macrophages. TPL‐2 therefore controls innate immune responses of macrophages to bacteria via V‐ATPase induction of phagosome maturation.


Introduction
A key component of the innate immune response involves the killing of phagocytosed microbes, such as bacteria, by macrophages (Pauwels et al, 2017). Phagocytosis internalises bacteria into membrane-bound vacuoles in the cytoplasm called phagosomes (Flannagan et al, 2012). The nascent phagosome is innocuous, and to kill internalised bacteria, the phagosome must mature via an ordered series of membrane fusion and fission events with endosomes and ultimately with lysosomes to form the phagolysosome, a potent microbicidal organelle (Flannagan et al, 2009). During the maturation process, phagosomes become increasingly acidic, highly oxidative and enriched with proteases and hydrolases that can degrade the internalised bacteria (Flannagan et al, 2009). Phagosome acidification is mediated by V-ATPases, ATP-dependent proton pumps, which are recruited to the phagosome during maturation (Lukacs et al, 1990;Sun-Wada et al, 2009). Acidification inhibits bacterial growth (Downey et al, 1999;Ip et al, 2010), activates cathepsin proteases that degrade internalised bacteria (Yates et al, 2005) and promotes NOX2 production of reactive oxygen species (ROS) that damage bacterial proteins, lipids and nucleic acids (Savina et al, 2006).
Bacterial infection also engages macrophage Toll-like receptors (TLRs) on the plasma and endosomal membranes, triggering intracellular signalling pathways that control activation of NF-jB transcription factors and each of the major mitogen-activated protein (MAP) kinases (extracellular signal-regulated kinases 1 and 2 [ERK1/2], Jun amino-terminal kinases 1 and 2 [JNK1/2] and p38a) (Arthur & Ley, 2013). Together, these stimulate the expression of multiple genes, the products of which can directly target the invading bacterium (e.g. antimicrobial peptides and NOS2) or induce recruitment of additional immune cells (e.g. cytokines and chemokines) (Smale, 2010;Kawasaki & Kawai, 2014). This inflammatory response is essential for killing of bacteria by macrophages and also the subsequent induction of the adaptive immune response (Nau et al, 2002).
TLR activation of ERK1/2 and p38a in macrophages is mediated by TPL-2 (tumour progression locus 2, also known as MAP3K8), a MAP 3-kinase that directly phosphorylates and activates the MAP 2kinases MKK1, 2, 3 and 6 Pattison et al, 2016). TPL-2 is critical for inflammatory immune responses to invading bacteria, fungi and viruses. TPL-2 forms a stoichiometric complex with NF-jB1 p105 and A20 binding inhibitor of NF-jB (ABIN)-2 in unstimulated macrophages (Lang et al, 2004). NF-jB1 p105 functions as an inhibitor of TPL-2 and TLR activation of ERK1/2, and p38a requires the liberation of TPL-2 from p105-mediated inhibition . This results from p105 proteolysis by the proteasome triggered by IjB kinase (IKK)-induced p105 phosphorylation (Belich et al, 1999). ABIN-2 is also released from p105 after TLR stimulation but is not required for TPL-2 activation, and its function in innate immune responses remains unclear.
TPL-2 expression is required for efficient immune responses to Citrobacter rodentium, decreasing the bacterial burden and dissemination to the liver and spleen (Acuff et al, 2017). TPL-2 deficiency reduces neutrophil recruitment to the colon at peak infection. In vitro experiments have shown that TPL-2 expression in neutrophils is required for LPS activation of ERK1/2 and induction of tumour necrosis factor (TNF) secretion, similar to that in macrophages (Acuff et al, 2017). TPL-2 is also required for efficient killing of phagocytosed C. rodentium, although phagocytic uptake of bacteria is TPL-2-independent (Acuff et al, 2017). However, the mechanism by which TPL-2 regulates bacterial killing has not been established. A recent study by one of our groups using quantitative mass spectrometry demonstrated that TPL-2 catalytic activity potentially regulates intracellular trafficking. Tpl2 D270A kinase-dead mutation reduced the phosphorylation of several proteins linked to endocytosis, vesicle trafficking and GTPase signalling in LPS-stimulated macrophages (Pattison et al, 2016). Together, these results raised the interesting possibility that TPL-2 promotes the killing of internalised bacteria by controlling phagosome maturation in myeloid cells.
In the present study, the potential regulation of phagosome maturation by TPL-2 in macrophages was investigated. TPL-2 catalytic activity was found to induce macrophage phagosome proteolytic activity and phagosome acidification, both independently of its ability to activate MAP kinase signalling. These effects were mediated by direct regulation of V-ATPase assembly and function via controlling the phosphorylation of the V-ATPase-associated regulatory protein DMXL1. Consistent with these findings, TPL-2 catalytic activity was necessary for efficient macrophage killing of phagocytosed Staphylococcus aureus and Citrobacter rodentium.
These results indicate that TPL-2 has two key functions in the innate immune response of macrophages to bacterial infection. TPL-2 mediates TLR activation of MAP kinase signalling to control inflammation by modulation of gene expression and directly promotes bacterial killing by MAP kinase-independent induction of phagosome acidification.
Next, a confocal microscopy assay was used to monitor cathepsin protease activation following phagocytic uptake of latex beads by Tpl2 D270A/D270A BMDMs compared to WT control cells, using a fluorescently labelled cathepsin L target peptide. Thirty mins after bead uptake, phagosome cathepsin activity was significantly reduced by Tpl2 D270A mutation ( Fig 1C). These results showed that TPL-2 catalytic activity is required to increase protease activity inside phagosomes of primary macrophages.
During maturation, the lumens of phagosomes become increasingly acidic due to the action of V-ATPases that are recruited by fusion of phagosomes initially with endocytic vesicles and ultimately lysosomes (Kinchen & Ravichandran, 2008). Intra-phagosomal acidification was assayed using latex beads coupled to the pH indicator BCECF (Russell et al, 1995). Real-time measurements showed that phagosomal acidification was significantly decreased in Tpl2 D270A/D270A BMDMs compared to WT BMDMs ( Fig 1D). Tpl2 D270A mutation reduced phagosome acidification to a similar degree to the pre-treatment of WT BMDMs with the V-ATPase inhibitor bafilomycin A1 (Fig 1D). Consistent with these results, analysis of phagosomal pH by staining with LysoTracker Red, a dye that labels acidic compartments, and confocal microscopy demonstrated that Tpl2 D270A mutation reduced LysoTracker Red staining following latex bead uptake ( Fig 1E).
The production of reactive oxygen species (ROS) in phagosomes by NAPDH oxidase is essential for killing internalised bacteria and is dependent on an intra-phagosomal proton gradient by V-ATPases (Mantegazza et al, 2008). Consistent with its inhibitory effects on phagosome acidification, Tpl2 D270A mutation significantly reduced ROS generation in BMDMs after uptake of latex beads coupled to the fluorescence reporter substrate OxyBURST Green BSA ( Fig 1F)  . Tpl2 D270A mutation also significantly reduced ROS generation detected microscopically using ROS Deep Red dye following latex bead uptake ( Fig 1G).
Together, these results show that TPL-2 catalytic activity promotes the maturation of phagosomes in macrophages.

TPL-2 induces phagosome maturation independently of MAP kinase activation
TPL-2's established functions in innate immunity are mediated by activation of MAP kinase pathways (Gantke et al, 2011). However, uptake of latex beads by BMDMs did not induce ERK1/2 and p38a activation, as detected by phospho-antibody immunoblotting, suggesting that TPL-2 modulated phagosome maturation independently of its ability to activate MAP kinases. Genetic and pharmacological experiments were used to investigate whether MAP kinase activation was required for TPL-2 catalytic activity to promote phagosome maturation.
IKK triggers proteolysis of NF-jB1 p105 by phosphorylating Ser930 and Ser935 in the p105 PEST region .
Average fluorescence intensity of the cathepsin probe per cell was quantified (n = 40-51 cells). D Intra-phagosomal acidification in WT and Tpl2[D270A] BMDMs was monitored following uptake of BCECF-coupled latex beads. BMDMs were pre-treated with 1 lM bafilomycin A1 for 15 min to inhibit V-ATPases (n = 4 wells). E pH assay in WT and Tpl2[D270A] BMDMs upon 0.5 h after incubation with latex beads. BMDMs were stained with the LysoTracker Red DND-99 dye (red). Average fluorescence intensity of the LysoTracker Red probe per cell was quantified (n = 95-126 cells). F Intra-phagosomal oxidative burst in WT and Tpl2[D270A] BMDMs was assayed following uptake of OxyBURST Green BSA / AF594 latex beads. BMDMs were pretreated with 1 lM bafilomycin A1 for 15 min to inhibit ROS production (n = 4 wells). G Reactive oxygen species assay in WT and Tpl2[D270A] BMDMs upon 0.5 h after incubation with latex beads. BMDMs were stained with the ROS Deep Red dye.
Average fluorescence intensity of the ROS Deep Red probe per cell was quantified (n = 80-110 cells).
Data information: One representative experiment out of three shown. Error bars and shaded areas represent SEM. ****P < 0.0001. Panels (B, D, F) Paired Mann-Whitney t-test; All differences relative to WT are ****. Panels (C, E, G) Student's unpaired t-test.

A B
Acidification (

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The EMBO Journal 40: e106188 | 2021 ª 2021 The Authors processes were reduced by Nfkb1 SSAA Tpl2 D270A compound mutation confirming their dependence on TPL-2 catalytic activity (Fig 2A  and B). Inhibition of ERK1/2 phosphorylation in Nfkb1 SSAA and Nfkb1 SSAA Tpl2 D270A BMDMs following LPS stimulation was confirmed by immunoblotting ( Fig 2C). In line with these genetic data, simultaneous pharmacological inhibition of ERK1/2 (PD0325901) and p38a (VX-745) activity did not alter phagosome proteolysis or acidification (Fig 2D and E). In contrast, TPL-2 inhibition with the small molecule inhibitor C34 ) significantly reduced both processes to a similar degree as Tpl2 D270A mutation (Fig 2D and E). Substantial reductions of MAP kinase activation by pharmacological inhibitors were confirmed by immunoblotting of LPS-stimulated BMDM lysates ( Fig 2F). TPL-2 catalytic activity therefore stimulated phagosome maturation in macrophages independently of MAP kinase activation (via unknown substrates). The inhibitory effects of C34 on phagosome proteolysis and acidification in WT BMDMs also demonstrated that the effects of TPL-2 catalytic activity on phagosome maturation were mediated acutely following latex bead uptake.
To investigate the role of TPL-2 in phagosome maturation in human primary macrophages, macrophages were generated from monocytes isolated from peripheral blood by culturing in GM-CSF and phagosome maturation was assayed as for BMDMs. C34 inhibition of TPL-2 catalytic activity reduced intra-phagosomal proteolysis ( Fig 2G) and acidification ( Fig 2H) in monocyte-derived human macrophages compared to cells pre-treated with vehicle control. Pharmacological inhibition of MEK1/2 (PD0325901) (Ciuffreda et al, 2009) and p38a (VX-745) (Duffy et al, 2011) did not alter either phagosome proteolysis (Fig 2I) or acidification (Fig 2J), although these inhibitors potently inhibited phosphorylation of ERK1/2 and p38a following LPS stimulation ( Fig 2K). These experiments showed that MAP kinase-independent stimulation of phagosome maturation by TPL-2 catalytic activity was conserved between mouse and human primary macrophages.

TPL-2 catalytic activity regulates the protein composition of phagosomes
To further study the molecular mechanisms by which TPL-2 kinase activity promotes phagosome maturation, the composition of phagosomes isolated from Tpl2 D270A/D270A and WT BMDMs following uptake of latex beads was analysed by mass spectrometry. Tpl2 D270A mutation significantly altered the composition of the phagosome proteome ( Fig 3A and B, Dataset EV1). Phagosome abundance of numerous Rab GTPases was decreased by Tpl2 D270A mutation ( Fig 3A). These included RAB5 and RAB7, which have key roles in regulating the maturation of phagosomes (Vieira et al, 2003;Huynh et al, 2007). The abundance of LAMP1, which is essential for late phagosome/lysosome fusion, was also decreased in Tpl2 D270A/D270A phagosomes compared with WT cells (Fig 3A and D). The levels of seven V-ATPase subunits, including V-ATPase subunits D, D1 and E1, were also significantly reduced ( Fig 3C). This finding was particularly interesting given the inhibitory effect of Tpl2 D270A mutation on phagosome acidification. Phagosome abundance of four members of the LAMTOR protein complex, a key regulator of lysosomal trafficking (Colaço & J€ a€ attel€ a, 2017), was also reduced in Tpl2 D270A/D270A BMDMs compared to controls. Immunoblotting confirmed that Tpl2 D270A mutation reduced levels of RAB5 GTPase and LAMP-1 associated with purified phagosomes (Fig 3D). The effect of TPL-2 catalytic activity on the abundance of phagosomal proteins was selective since abundance of numerous proteins on phagosomes, including vimentin, SNARE, vacuolar protein sorting (VPS) and ESCRT proteins, was similar between WT and Tpl2 D270A/D270A BMDMs (Figs 3D and EV1).
Consistent with these changes in phagosome composition, gene set enrichment analyses to identify significantly downregulated biological processes revealed that Tpl2 D270A mutation decreased the abundance of proteins involved in vesicle-mediated transport, receptor-mediated transport, ion transport, vesicle proteolysis and regulation of pH ( Fig 3B). These processes are all important in the generation of mature phagolysosomes (Flannagan et al, 2009;Pauwels et al, 2017).
Together, these experiments indicated that blocking TPL-2 catalytic activity substantially changed the composition of phagosomes in BMDMs, reducing the abundance of several proteins with key roles in regulating different stages of phagosome maturation. Importantly, this analysis revealed that TPL-2 catalytic activity promoted the recruitment of V-ATPase subunits to phagosomes during their maturation suggesting a possible mechanism for TPL-2 control of phagosome acidification. BMDMs were immunoblotted for the indicated antigens. 1 lM bafilomycin A1 was added to inhibit V-ATPases. G-K Experiments were performed using human primary monocyte-derived macrophages. (G) Intra-phagosomal proteolysis in human macrophages was monitored following uptake of DQ Green BSA / AF594 latex beads. TPL-2 catalytic kinase activity was blocked by pre-treatment with 10 lM C34 for 1 h (n = 4 wells). (H) Intra-phagosomal acidification in human macrophages was monitored following uptake of BCECF-coupled latex beads (n = 4 wells). (I) Intra-phagosomal proteolysis in human macrophages was assayed as in Fig  Data information: Data were analysed by Student's t-test. Error bars represent SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

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The EMBO    To further investigate the mechanism by which TPL-2 kinase activity regulated phagosome maturation, the phosphoproteome in Tpl2 D270A/D270A and WT BMDMs was characterised 30 min after phagocytic uptake of latex beads. TMT (tandem mass tag) labelling and mass spectrometry were used to quantify differences in phosphopeptide abundance (Dataset EV2). The phospho-site most highly downregulated by Tpl2 D270A mutation was Ser141 on TPL-2 itself (Fig 4A), a known autophosphorylation site (Stafford et al, 2006;Xu et al, 2018). Two of the next most downregulated phospho-sites were Ser1903 and Ser1904 of DMXL1, which were both reduced over threefold. Phosphorylation of DMXL1 on Ser434, Ser915, Thr1373 and Ser1900 was not altered by Tpl2 D270A mutation indicating that TPL-2 catalytic activity did not change DMXL1 protein abundance (Fig 4A).
A phospho-Ser1903 DMXL1-peptide antibody was generated and immunoblotting was used to validate the MS results. This analysis demonstrated that phagocytic uptake of latex beads by WT BMDMs induced the phosphorylation of DMXL1 on Ser1903, and this was blocked in Tpl2 D270A/D270A BMDMs (Fig 4B). A recent study demonstrated that DMXL1, a WD40 repeat protein, interacts with the V-ATPase complex and is required for acidification of endocytic vesicles (Merkulova et al, 2015). Together, these results raised the interesting possibility that TPL-2 might promote phagosome acidification by inducing the phosphorylation of DMXL1.
The role of DMXL1 in intracellular vesicle acidification was previously studied in a kidney cell line by siRNA knockdown (Merkulova et al, 2015). Immortalised BMDMs (iBMDMs) from WT C57BL/6 and Tpl2 D270A/D270A mice were used to investigate whether DMXL1 was required for phagosome maturation in macrophages. Initial experiments determined whether TPL-2 catalytic activity was required for phagosome maturation in iBMDMs similar to primary BMDMs. This was monitored following uptake of fluorescently labelled latex beads, initially by testing the effect of blocking TPL-2 catalytic activity in WT iBMDMs pharmacologically. C34 TPL-2 inhibitor significantly reduced phagosome proteolysis ( Fig EV2A) and acidification ( Fig EV2B). Importantly, phagosome proteolysis ( Fig EV2A) and acidification ( Fig EV2B) were also significantly impaired in Tpl2 D270A/D270A iBMDMs relative to WT iBMDMs. These results showed that TPL-2 catalytic activity promoted phagosome maturation in immortalised macrophages, similar to primary BMDMs.
To investigate whether DMXL1 was required for phagosome acidification, Dmxl1 mRNA in WT iBMDMs was depleted by over 90% using Dmxl1 siRNA oligonucleotides ( Fig 4C). Following uptake of BCECF latex beads, phagosome acidification was significantly reduced compared to WT iBMDMs transfected with nontargeting siRNAs (Fig 4D). DMXL1 depletion also decreased phagosome proteolysis monitored using DQ green BSA latex beads. (Fig EV3A). These results demonstrated that DMXL1 expression was required for phagosome acidification in macrophages. The observation that DMXL1 expression was also required for phagosome proteolysis was consistent with the known role of V-ATPasemediated acidification for the induction of proteolytic activity in phagosomes (Lennon-Dum enil et al, 2002;Kinchen & Ravichandran, 2008). Depletion of DMXL2, a paralog of DMXL1, did not alter phagosome acidification or proteolysis in macrophages (Fig EV3B-D).
To study whether TPL-2 and DMXL1 function in the same pathway, the effect of DMXL1 depletion on phagosome maturation was monitored in Tpl2 D270A/D270A iBMDMs. DMXL1 siRNA knockdown did not alter phagosome acidification (Fig 4E) relative to transfection with a non-targeting siRNA pool. Similarly, DMXL1 depletion in Tpl2 D270A/D270A iBMDMs did not alter phagosomal proteolysis compared to Tpl2 D270A/D270A iBMDMs transfected with a non-targeting siRNA pool (Fig EV3E). These results were consistent with TPL-2 catalytic activity promoting phagosome acidification and proteolysis via DMXL1.
Together, the results in this section suggest that TPL-2 stimulates phagosome acidification in macrophages by regulating the phosphorylation of DMXL1 to control the assembly of active V-ATPase complexes.

TPL-2 catalytic activity induces phagosome maturation following phagocytic uptake of Staphylococcus aureus
Phagocytosis is a critical component of the innate immune response to bacterial infection. To investigate whether TPL-2 regulates phagosome maturation following phagocytic uptake of bacteria, BMDMs were infected with S. aureus. Tpl2 D270A mutation did not alter the phagocytic uptake of YFP-labelled S. aureus (Fig 5A), consistent with results using latex beads. However, the total levels of active cathepsin per YFP + cell were significantly reduced, as detected using a labelled cathepsin L peptide substrate and confocal microscopy (Fig 5B and C). Furthermore, the percentages of cathepsin L-positive S. aureus phagosomes in Tpl2 D270A/D270A BMDMs were decreased approximately five-fold compared to WT BMDMs (Fig 5D).
Phagosome acidification was also analysed following S. aureus uptake by LysoTracker Red staining and confocal microscopy. Tpl2 D270A mutation decreased the fluorescence intensity for Lyso-Tracker Red per YFP + cell (Fig 5E and F). The percentage of LysoTracker Red + phagosomes containing S. aureus was also significantly reduced (Fig 5G). The effect of TPL-2 catalytic activity on phagosome maturation was also investigated using specific markers phagosome maturation. Co-localisation of YFP + S. aureus with the early endosomal antigen 1 (EEA1), a marker of early phagosomes, was reduced in Tpl2 D270A/D270A BMDMs compared to WT cells (Fig 5H and I). Recruitment of LAMP-1, a marker for late phagosomes and phagolysosomes, to YFP + S. aureus phagosomes was also reduced by Tpl2 D270A mutation (Fig 5J and K).
Together, these results indicated that TPL-2 catalytic activity in macrophages promotes maturation of phagosomes containing internalised S. aureus, stimulating phagosome acidification and cathepsin activation. These results confirmed our earlier findings monitoring the effects of TPL-2 signalling on phagosome maturation in macrophages following uptake of latex beads.
Tpl2 D270A mutation impairs killing of phagocytosed bacteria Following engulfment, bacterial killing by macrophages is dependent on the maturation of phagosomes to generate phagolysosomes that have microbicidal activity. To investigate whether Tpl2 D270A mutation impaired the killing of phagocytosed bacteria, BMDMs were infected with S. aureus and the bacterial load was measured 8h after infection by enumerating CFUs.
Bacterial killing was significantly reduced in Tpl2 D270A/D270A BMDMs compared to WT cells (Fig 6A). Consistent with this, the fluorescence intensity of YFP + S. aureus was higher in mutant cells compared to WT controls ( Fig 6B). Importantly, the degree by which Tpl2 D270A mutation impaired bacterial killing was comparable to that detected by treatment of WT BMDMs with the V-ATPase inhibitor bafilomycin A1 (Fig 6A), which has pronounced inhibitory effects on phagosome maturation (Yoshimori et al, 1991;Bidani et al, 2000).
The partial inhibitory effect of bafilomycin A1 on bacterial killing implied that BMDMs also employed a phagosome-independent mechanism to kill S. aureus. This was likely due to the escape of bacteria from phagosomes and/or injection of poreforming toxins into the cytosol, which result in activation of inflammasomes and induction of pyroptosis (Seilie & Wardenburg, 2017). Consistent with this, S. aureus infection decreased the viability of BMDMs (Fig 6C) but this was not altered by Tpl2 D270A mutation, as indicated by comparable levels of released lactate dehydrogenase ( Fig 6C). Furthermore, gasdermin D p30 immunoblotting demonstrated that S. aureus infection induced Data information: One representative experiment out of three shown. Error bars represent SEM. P < 0.01, ****P < 0.0001. Student's unpaired t-test.

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The EMBO Journal 40: e106188 | 2021 ª 2021 The Authors similar levels of pyroptosis in Tpl2 D270A/D270A and WT cells ( Fig 6D). These data show that decreased bacterial killing in Tpl2 D270A/D270A BMDMs compared to controls is not due to changes in host cell death and that pyroptosis of BMDMs induced by S. aureus infection is independent of TPL-2 catalytic activity.
To determine whether TPL-2 catalytic activity promoted the killing of Gram-negative bacteria, BMDMs were infected with Citrobacter rodentium. Phagocytic uptake of bacteria was similar between Tpl2 D270A/D270A and WT BMDMs (Fig 6E). Measurement of the bacterial load at 2 h showed that Tpl2 D270A mutation impaired the killing of C. rodentium, to a similar extent as bafilomycin A1 treatment ( Fig 6E). Acidification of phagosomes containing GFP-C. rodentium was monitored by confocal microscopy and Lyso-Tracker Red staining. Tpl2 D270A mutation significantly decreased the percentage of LysoTracker Red + phagosomes containing C. rodentium (Fig 6F and G). TPL-2 catalytic activity is therefore required for the acidification of phagosomes containing Gram-positive or Gram-negative bacteria following infection of macrophages and for efficient killing of these bacteria.

Discussion
Macrophage killing of phagocytosed bacteria is critical for controlling bacterial infections in the initial phase of an innate immune response. The recruitment of V-ATPases to phagosomes during their maturation is required for phagosome acidification, which is essential for further maturation to generate microbicidal phagolysosomes containing active cathepsin proteases (Kinchen & Ravichandran, 2008). In this study, genetic and pharmacological experiments demonstrated that TPL-2 is a positive regulator of phagosome acidification and proteolytic activity in mouse and human primary macrophages after uptake of latex beads. Consistent with this, following infection of macrophages by S. aureus, TPL-2 kinase activity stimulates acidification and activation of cathepsins in phagosomes containing internalised bacteria, promoting their efficient killing.
The stimulation of MAP kinase signalling in myeloid cells by TPL-2 during innate immune responses is well established . TPL-2 is essential for activation of ERK1/2 MAP kinases by multiple TLRs in macrophages (Banerjee et al, 2006) and also contributes to TLR activation of p38a MAP kinase (Pattison et al, 2016). However, pharmacological and genetic experiments herein showed that TPL-2 stimulated phagosome acidification and proteolysis independently of MAP kinase activation. Phosphoproteomic analyses identified that TPL-2 catalytic activity is required for Ser1903 and Ser1904 phosphorylation of DMXL1 in macrophages following phagocytic uptake of latex beads. DMXL1 is a recently identified V-ATPase interacting protein that regulates V-ATPasemediated acidification of endocytic vesicles in a kidney cell line (Merkulova et al, 2015). Knockdown experiments demonstrated that DMXL1 was also required for phagosome acidification (and proteolysis) in macrophages after latex bead internalisation, while DMXL2 was dispensable. Expression of a DMXL1 fragment containing the Ser1903/Ser1904 phosphorylation sites rescued the acidification defect in DMXL1-deficient WT macrophages but not DMXL1deficient Tpl2 D270A/D270A macrophages. Together, these results indicate that TPL-2 induced phosphorylation of DMXL1 Ser1903 and Ser1904 promotes V-ATPase-mediated acidification of phagosomes.
V-ATPases consist of a peripheral membrane subcomplex called V1, which contains the sites of ATP hydrolysis, associated with an integral membrane subcomplex called V0, which encompasses the proton pore. Initial studies in yeast showed that V-ATPases are regulated by the reversible interaction of V0 and V1 subcomplexes; release of V1 into the cytosol inhibits both ATPase and proton transport activities (Parra & Kane, 1998). DMXL1 is homologous to DMXL2 (also known as Rabconnectin-3) that binds to the cytosolic V1 subcomplex of V-ATPases and promotes its association with membrane-bound V0 V-ATPase subcomplex to form an active V0/ V1 holoenzyme following Notch stimulation (Nagano et al, 2002;Sethi et al, 2010). Our results show that TPL-2 catalytic activity promotes V0/V1 association following uptake of latex beads by macrophages, suggesting that TPL-2 stimulates phagosome acidification by inducing the assembly of active V-ATPase complexes via control of DMXL1 phosphorylation.
A phospho-Ser1903 DMXL1 antibody showed that the phosphorylation of DMXL1 is induced by TPL-2 following macrophage uptake of latex beads. This implies that scavenger receptors involved in phagocytosis of latex beads (Sulahian et al, 2008) trigger a TPL-2-dependent signalling pathway that leads to Ser1903 phosphorylation of DMXL1. In future studies, it will be important to determine whether DMXL1 Ser1903 is phosphorylated directly by TPL-2 itself or a TPL-2-regulated kinase. The failure of Nfkb1 SSAA mutation to block TPL-2 induction of phagosome maturation raises the possibility that this pathway induces DMXL1 phosphorylation while TPL-2 still complexed with NF-jB1 p105 and ABIN-2 (Yang et al, 2012;Pattison et al, 2016). However, it cannot be ruled out that phosphorylation of p105 on sites other than those targeted by the IKK complex induces p105 proteolysis following phagocytic uptake of latex beads, releasing TPL-2 from p105 to control DMXL1 phosphorylation.
Killing of S. aureus, measured by CFU assay, showed that Tpl2 D270A mutation reduced the efficiency of BMDM killing of internalised bacteria. This correlated with reduced acidification and cathepsin activation in phagosomes containing fluorescently ◀ Figure 6. Tpl2[D270A] mutation impairs macrophage killing of S. aureus.
A BMDMs of the indicated genotypes were infected with YFP-S. aureus (MOI 10) for 0.5 h. S. aureus CFU were assessed at 8 h after infection and normalised to CFU at 1 h (n = 12; 2 biological and 6 technical replicates per condition per experiment). As a control, WT cells were treated with bafilomycin A to block V-ATPase acidification of phagosomes and phagosome proteolysis. B Quantification of YFP-S. aureus average fluorescence intensity per cell from Fig 5 panel (A) (n = 66-85 cells). C Cell death following was assayed by measuring LDH release from cytosol of BMDMs 8 and 24 h post-infection with S. aureus (MOI 10; n = 4 wells). D Whole cell extracts from WT and Tpl2[D270A] BMDMs following S. aureus infection (MOI 10) were immunoblotted for the gasdermin D p30 cleavage product. HSP90 was used as a loading control. E BMDMs of the indicated genotypes were infected with GFP-C. rodentium (MOI 2) for 0.5 h. C. rodentium CFU were assessed at 2 h after infection and normalised to CFU at 0.5 h (n = 12; 2 biological and 6 technical replicates per condition per experiment). As a control, WT cells were treated with bafilomycin A. F Representative images of GFP + BMDMs labelled with LysoTracker Red DND-99 dye (red) and DAPI, 1 h post-infection with GFP-C. rodentium (green). G Quantification of LysoTracker Red co-localisation with GFP-C. rodentium from panel (F) (n = 54-63 cells).

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The EMBO Journal 40: e106188 | 2021 ª 2021 The Authors labelled bacteria. In addition, a decreased fraction of phagosomes containing fluorescently labelled bacteria stained positive for the early and late markers for phagosome maturation, EEA1 and LAMP-1, respectively. Together, these results showed that Tpl2 D270A mutation impaired the maturation of phagosomes containing internalised S. aureus bacteria, which explained the reduced efficiency of bacterial killing in Tpl2 D270A/D270A BMDMs compared to WT controls. TPL-2 catalytic activity was also required for acidification of C. rodentium phagosomes and efficient killing of C. rodentium suggesting that TPL-2-dependent induction of phagosome maturation is important in innate immune responses to Gram-positive and Gram-negative bacterial species. The inhibitory effect of Tpl2 D270A mutation on S. aureus killing was of a similar magnitude to that observed with WT cell treatment with bafilomycin A1, which directly inhibited V-ATPase function. Since bafilomycin A1 has pronounced inhibitory effects on phagosome maturation and endosomal trafficking (Yamamoto et al, 1998), these experiments showed that in vitro killing of S. aureus by BMDMs was only partially mediated by phagolysosomes. In both WT and Tpl2 D270A/D270A BMDMs, the phagosome-independent killing of internalised bacteria was likely mediated by inflammasome activation and pyroptosis induced by escape of bacteria into the cytosol or bacterial toxins (Man et al, 2017). Assay of cell death (LDH release) and gasdermin D cleavage demonstrated that Tpl2 D270A mutation did not alter pyroptosis induced by S. aureus infection of macrophages (Aglietti et al, 2016). Cleaved gasdermin D has been shown to directly kill both intracellular and extracellular bacteria . Staphylococcus aureus released from permeabilised cells was also exposed to bactericidal gentamicin in the culture medium. Together, our experiments show that decreased phagosome-mediated killing does not prevent S. aureus-induced pyroptosis and that host cell death cannot explain the effects of Tpl2 D270A mutation on killing of S. aureus.
TPL-2, therefore, has two key roles in the innate immune response of macrophages to bacterial infection. TPL-2 activation of MAP kinase pathways, triggered by TLRs at the cell surface, and, independently of MAP kinase activation, TPL-2 stimulates V-ATPase-mediated acidification of phagosomes to promote more efficient bacterial killing in phagolysosomes. These processes function together to limit bacterial replication in innate immune responses.

Mice
Mouse strains were bred in a specific-pathogen-free environment at the Francis Crick Institute (London, United Kingdom), and all experiments carried out in accordance with regulations of the UK Home Office. Map3k8 D270A/D270A , Nfkb1 SSAA/SSAA , and Nfkb1 SSAA/SSAA Map3k8 D270A/D270A have been described previously (Sriskantharajah et al, 2009(Sriskantharajah et al, , 2014. All mouse strains were fully backcrossed on to a C57BL/6Jax background (The Jackson Laboratory).

Human monocyte-derived macrophages
Blood from healthy donors was used in this study, which was approved by the NHS Health Research Authority and complied with ª 2021 The Authors The EMBO Journal 40: e106188 | 2021 the Human Tissue Act 2004. Human peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats provided by NHS Blood and Transplant Colindale. PBMCs were isolated from peripheral blood by density gradient centrifugation using Ficoll-Paque Plus (GE Healthcare). PBMCs were labelled with CD14 MicroBeads (MACS Miltenyi Biotec). CD14 + PBMCs were applied to, washed and eluted from LS columns (MACS Miltenyi Biotec) according to manufacturer instructions. CD14 + PBMCs were seeded at 2 × 10 7 cells per 140 mm Nunc dish (Sigma-Aldrich) in RPMI-1640 medium containing 10% FBS, 10 mM HEPES, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 lg/ml streptomycin, 2 mM L-glutamine and 50 ng/ml human GM-CSF (STEMCELL Technologies). Cells were fed on day 4 and resulting macrophages harvested on day 7.

Phagosome proteolysis assay
Bead coupling procedures and proteolysis assays were adapted from protocols developed by the Valenzeno and Russell laboratories (Russell et al, 1995;Yates et al, 2005;Yates & Russell, 2008). DQ Green BSA (Thermo Fisher Scientific) and Alexa Fluor 594 succinimidyl ester (Thermo Fisher Scientific) were coupled to carboxylated 3 µm silica beads (Kisker Biotech), as described (Yates et al, 2005;Yates & Russell, 2008). BMDMs were seeded on 96-well plates (Greiner CELLSTARâ) at 1 × 10 5 cells/well and cultured overnight. DQ Green BSA/Alexa Fluor 594-coupled silica beads were diluted 1:300 in 5% FBS in DPBS and incubated with cells for 3 min at RT. External beads were removed by washing with pre-warmed 5% FBS in DPBS, and real-time fluorescence intensities of cells were measured at ex/em 490/520 nm and at ex/em 590/620 nm at 37°C using a CLARIOstar (BMG Labtech) reader at 2min intervals over 6 h. The calibration fluorophore Alexa Fluor 594 was used to normalise against phagocytic bead uptake. Plots were generated from DQ Green BSA to Alexa Fluor 594 ratios.

Oxidative burst assay
OxyBURST Green H2HFF BSA (Thermo Fisher Scientific) and Alexa Fluor 594 succinimidyl ester (Thermo Fisher Scientific) were coupled to carboxylated 3 µm silica beads (Kisker Biotech), as described . OxyBURST Green BSA/Alexa Fluor 594-coupled silica beads were diluted 1:200 in 5% FBS in DPBS and incubated with BMDMs for 3 min at RT. External beads were removed by washing and replaced with pre-warmed 5% FBS in DPBS. Real-time fluorescence intensities of cells were measured at ex/em 480/520 nm and at ex/em 590/620 nm at 37°C using a CLARIOstar (BMG Labtech) reader at 2 min intervals over 90 min. Oxidation of H2HFF-OxyBURST generates a fluorescein-based product, which emits a signal at 520 nm upon excitation at 480 nm ).

Phagosome isolation
Phagosomes were isolated from BMDMs as previously described (Trost et al, 2009;Dill et al, 2015) Briefly, 0.93 µM blue-dyed carboxylated polystyrene beads (Estapor Merck Chimie SAS) were diluted 1:50 in complete RPMI-1640 medium (without L929 cell supernatant) and induced for 30 min with 1 × 10 7 BMDMs per 10 cm Nunc dish. BMDMs were washed and lysed in hypotonic buffer by Dounce homogenisation. Nuclei and cell debris were removed by centrifugation 1,000 ×g for 5 min at 4°C. Phagosomes were then isolated using a discontinuous sucrose gradient. Phagosome fractions were separated in a SW41Ti swinging bucket rotor in an Optima TM L-90 K ultracentrifuge (Beckman Coulter) at 72,300 ×g for 1 h at 4°C. Blue-coloured latex bead-containing phagosomes were collected from the interface of the 10 and 25% sucrose fractions.

Proteomic sample preparation of phagosomes
Isolated phagosomes were lysed in 5% SDS, 50 mM triethylammonium bicarbonate (TEAB) pH 7.5 (S-Trap), sonicated and protein concentration determined using the BCA Protein Assay Kit (Pierce Protein). Samples were reduced in 10 mM tris(2-carboxyethyl)phosphine for 30 min at RT followed by alkylation in 10 mM iodoacetamide for 30 min. Samples were acidified and digested on an S-trap spin column by addition of porcine trypsin at 1:10 (Pierce) for 2 h at 47°C. Peptides were eluted in 50 mM TEAB pH 8.0, 0.2% formic acid and 0.2% formic acid in 50% acetonitrile, respectively.

Label-free MS acquisition of phagosome proteomes
Peptide samples were separated on an Ultimate 3000 RSLC system (Thermo Scientific) with a C18 PepMap, serving as a trapping column (2 cm × 100 µm ID, PepMap C18, 5 µm particles, 100 A pore size) followed by a 50 cm EASY-Spray column (50 cm × 75 µm ID, PepMap C18, 2 µm particles, 100 A pore size) (Thermo Scientific). Buffer A contained 0.1% formic acid and Buffer B 80% acetonitrile, 0.1% formic acid. Peptides were separated with a linear gradient of 1-35% (Buffer B) over 120 min followed by a step from 35 to 90% acetonitrile, 0.1% formic acid in 0.5 min at 300 nl/min and held at 90% for 4 min. The gradient was then decreased to 1% Buffer B in 0.5 min at 300 nl/min for 10 min. Mass spectrometric identification was performed on an Orbitrap QE HF mass spectrometer (Thermo Scientific) operated "TopN" datadependent mode in positive ion mode. FullScan spectra were acquired in a range from 400 m/z to 1,500 m/z, at a resolution of 120,000 (at 200 m/z), with an automated gain control (AGC) of 1 × 10 6 and a maximum injection time of 50 ms. Charge state screening is enabled to exclude precursors with a charge state of 1. For MS/MS fragmentation, the minimum AGC was set to 5,000 and the most intense precursor ions were isolated with a quadrupole mass filter width of 1.6 m/z and 0.5 m/z offset. Precursors were subjected to higher-energy collisional dissociation (HCD) fragmentation that was performed in one-step collision energy of 25%. MS/ MS fragments ions were analysed in the Orbitrap mass analyser with a 15,000 resolution at 200 m/z.

Sample preparation for Tandem Mass Tag (TMT) phosphoproteome analysis of BMDMs
BMDMs from WT or Tpl2 D270A/D270A mice were incubated with carboxylated polystyrene beads as for phagosome isolations. Protein content was estimated by protein assay, and a 200 lg aliquot of each sample was taken for further processing. Samples were reduced with 10 mM dithiothreitol for 25 min at 56°C and then alkylated with 20 mM iodoacetamide for 30 min at RT. The alkylation reaction was quenched with an additional 10 mM dithiothreitol, and then, each sample was diluted with 50 mM HEPES to reduce the urea concentration to < 2 M prior to digestion. Proteolytic digestion was carried out by the addition of 4 lg LysC (FUJIFILM Wako Pure Chemical Corporation) and incubated at 37°C for 2.5 h followed by the addition of 10 lg trypsin (Pierce) and overnight incubation at 37°C. After acidification, C18 MacroSpin columns (Nest Group) were used to clean up the digested peptide solutions and the eluted peptides dried by vacuum centrifugation. Samples were resuspended in 50 mM HEPES and labelled using the 0.8 mg Tandem Mass Tag 10plex isobaric reagent kit (Thermo Scientific) resuspended in acetonitrile. Labelling reactions were quenched with hydroxylamine, and a pool was made containing all of the samples. Acetonitrile content was removed from the pooled TMT solution by vacuum centrifugation and then acidified before using a Sep-Pak C18 (Waters) to clean up the labelled peptide pool prior to phosphopeptide enrichment. The eluted TMT-labelled peptides were dried by vacuum centrifugation, and phosphopeptide enrichment was subsequently carried out using the sequential metal oxide affinity chromatography (SMOAC) strategy with High Select TiO2 and Fe-NTA enrichment kits (Thermo Scientific). Eluates were immediately dried by vacuum centrifugation after enrichment and then combined prior to fractionation with the Pierce High pH Reversed-Phase Peptide Fractionation Kit (Thermo Scientific). All fractions were dried by vacuum centrifugation and stored at À80°C until the day of MS analysis.

MS data acquisition and processing for TMT phosphoproteome
Dried TMT-labelled phosphopeptide fractions were resuspended in 0.1% trifluoroacetic acid and then analysed by online nanoflow LC-MS/MS using an Orbitrap Fusion Lumos mass spectrometer (Thermo Scientific) coupled to an Ultimate 3000 RSLCnano (Thermo Scientific

Immunofluorescence
BMDMs were seeded on 13 × 1.5 mm round coverslips (VWR International) at 1 × 10 5 cells in a 24-well plate and rested overnight. Cells were treated as described in figure legends. After treatments, BMDMs were washed in PBS and fixed in eBioscience TM IC fixation buffer (Thermo Fisher Scientific) for 10 min at RT. BMDMs were permeabilised in 0.1% Triton X-100 in PBS for 4 min, washed and then blocked in 0.4% gelatin from cold water fish skin (Sigma-Aldrich) in PBS for 10 min. Following incubation in 0.1% sodium borohydride pH 8.0 (Sigma-Aldrich) for 10 min, cells were stained with primary antibody solution ( Cathepsin activity assay Following treatment, BMDMs cultured on coverslips (as described above) were washed twice with DPBS and then incubated with the Magic Red cathepsin L substrate (ICT942, Bio-Rad Antibodies) in 5% FBS in DPBS at a final dilution of 1:250 for 1 h at 37°C. Following washing, BMDMs were fixed, stained with DAPI and imaged as detailed above. Fluorescence of the Magic Red cathepsin L substrate was monitored at ex/em 592/628 nm.

LysoTracker red
Following treatment, BMDMs cultured on coverslips were washed twice with DPBS and then incubated with LysoTracker Red DND-99 (100nM final concentration; L7528, Thermo Fisher Scientific) in DPBS + 5% FBS for 1 h at 37°C. The immunostaining protocol was performed as described above, and fluorescence of LysoTracker Red was monitored at ex/em 577/590 nm. LysoTracker Red is an acidotropic red-fluorescent-dye, which stains acidic cellular compartments.

ROS assay
Following treatment, BMDMs cultured on coverslips were washed twice with DPBS and then incubated with ROS Deep Red dye (100 nM final concentration; ab186029, Abcam) in DPBS + 5% FBS for 1 h at 37°C. The immunostaining protocol was performed as described above, and fluorescence of ROS Deep Red dye was monitored at ex/em 650/675 nm. ROS Deep Red is a cell permeable Deep Red-fluorescent dye, which reacts with superoxides and hydroxyl radicals.

Image analysis
Images were analysed using ImageJ (Fiji) version 2.0.0 (National Institutes of Health, USA). Fluorescence intensities per BMDM were quantified by creating masks of cell outlines and determining the integrated density (IntDen). Ratios of integrated densities for two different markers in the same masked area were used to determine relative fluorescence intensity. Coloc 2, a Fiji plugin, was used to quantify co-localisation. To calculate Pearson's correlation coefficients and Manders' coefficients, Coloc 2 parameters were set to bisectional threshold regression with a point spread function of 3.0 and Costes randomisations of 100. Coefficients above the Coloc 2 autothreshold were selected for data analysis. For LAMP-1 staining, S. aureus bacteria were automatically counted by normalising the YFP fluorescence threshold across all fields of view and counting particles > 0.2 µm. Percentages of YFP-S. aureus associated with LAMP-1 rings were determined by analysing > 100 BMDMs including > 600 S. aureus bacteria per genotype from at least 40 random fields of view in three independent experiments.
siRNA-mediated knockdown of Dmxl1 iBMDMs were seeded at 5 × 10 4 cells/well in a tissue culture-treated Nunc 24-well plate and at 1 × 10 4 cells/well in a 96-well plate (Greiner CELLSTARâ) in complete RPMI-1640 containing 10% L929 cell supernatant and cultured overnight at 37°C. All siRNA stocks were reconstituted in siRNA buffer (Horizon Discovery) to 20 lM.
Quantitative real-time PCR (qRT-PCR) Following siRNA knockdown, iBMDMs were lysed in 350 ll Qiagen RLT lysis buffer (Thermo Fisher Scientific) containing b-mercaptoethanol at 1:100 (v/v). RNA was isolated using the Qiagen RNeasy Mini Kit (Thermo Fisher Scientific) and Qiagen RNase-free DNase set (Thermo Fisher Scientific), following manufacturer instructions. Isolated RNA was reverse transcribed to cDNA using the SuperScript VILO cDNA Synthesis Kit (Invitrogen). qRT-PCR was prepared in MicroAmp Optical 384-well plates (Applied Biosystems) with a total reaction volume of 7 ll, which contained 2 ll cDNA, 3.5 ll TaqMan Universal PCR Master Mix (Applied Biosystems), 0.35 ll TaqMan FAM-coupled probe and 1.15 ll nuclease-free water. TaqMan FAM-MGB probe Mm01261785_m1 was used for amplification of Dmxl1 (Thermo Fisher Scientific). qRT-PCR was performed on a QuantStudio 3 and 5 (Applied Biosystems) set to comparative C T mode, and Hprt (Mm01545399_m1) was used as a housekeeping gene for expression level normalisation.

SDS-PAGE and immunoblotting
Macrophages were lysed in 50 mM Tris-HCl pH 7.5, 150 mM sodium chloride, 1% Triton X-100, 10 mM sodium fluoride, 1 mM sodium pyrophosphate, 10 mM b-glycerophosphate, 2 mM EDTA, 100 lM sodium orthovanadate, 10% glycerol, supplemented with one EDTA-free protease inhibitor cocktail tablet (Roche). Protein concentration in cleared cell extracts was quantified using the Coomassie Protein Assay Kit (Pierce). Lysates were mixed with 5X sample buffer (250 mM Tris-HCl pH 6.8, 32.5% glycerol, 5% SDS, 5% b-mercaptoethanol) and heated for 10 min at 90°C. Samples were subjected to SDS-PAGE on a 10% acrylamide gel. After electrophoresis, proteins were transferred onto PVDF membranes (Bio-Rad Laboratories) using the Blot Turbo Transfer System (Bio-Rad Laboratories). Membranes were blocked with 5% (w/v) milk powder in PBS plus 0.05% Tween-20 for 1 h. Membranes were incubated with primary antibodies overnight at 4°C, washed four times in PBS plus 0.05% Tween-20 and then incubated with horseradish-peroxidase (HRP)-conjugated secondary antibodies. After four PBS plus 0.05% Tween-20 washes, membranes were incubated with Amersham ECL Western Blotting Detection Reagent (GE Healthcare Life Sciences) or Immobilon Western Chemiluminescent HRP Substrate (Millipore) to visualise protein levels following exposure of X-ray film (Scientific Laboratory Supplies).