Cellular Immunology 279 (2012) 46–52

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Cellular Immunology

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E3 ubiquitin ligase NKLAM is a macrophage phagosome protein and playsa role in bacterial killing

Donald W. Lawrence a, Jacki Kornbluth a,b,⇑a Department of Pathology, Saint Louis University School of Medicine, St. Louis, MO 63104, United Statesb Veterans Administration Medical Center, St. Louis, MO 63106, United States

a r t i c l e i n f o a b s t r a c t

Article history:Received 6 August 2012Accepted 12 September 2012Available online 1 October 2012

Keywords:MacrophageInnate immunityPhagocytosisPhagosome maturationKilling

0008-8749/$ - see front matter Published by Elsevierhttp://dx.doi.org/10.1016/j.cellimm.2012.09.004

Abbreviations: NKLAM, Natural Killer Lytic-Associmarrow-derived macrophage; TLR, Toll-like receptor;natural killer; RBR, RING in between RING; MFI, mea⇑ Corresponding author. Address: Department o

Research Center, Saint Louis University School of MBlvd., St. Louis, MO 63104, United States. Fax: +1 314

E-mail address: kornblut@slu.edu (J. Kornbluth).

Macrophages are a critically important component of the innate and adaptive immune systems. They areequipped with oxidative and non-oxidative mechanisms to kill ingested pathogens. Natural KillerLytic-Associated Molecule (NKLAM) is an E3 ubiquitin ligase expressed in macrophages and natural killercells. We show that NKLAM expression in macrophages was enhanced by Toll-like receptor agonists andpro-inflammatory cytokines. Using confocal microscopy, we found that NKLAM colocalized with ingestedEscherichia coli. In assays using IgG-opsonized latex beads as targets, we demonstrated that NKLAMtranslocated to the phagosome early during maturation at a time that coincided with elevated levelsof ubiquitinated phagosome proteins. In killing assays with bone marrow-derived macrophages fromwild type and NKLAM-deficient mice, we found that NKLAM-deficient macrophages demonstrated lesskilling of E. coli than wild type macrophages. Collectively, our data show that NKLAM is a novel compo-nent of macrophage phagosomes and is involved in macrophage bactericidal functions.

Published by Elsevier Inc.

1. Introduction to target proteins to the proteasome for degradation [5]. However,

Macrophages are a critical component of the innate and adap-tive immune system and act as a first line of defense against patho-gens. Pathogen binding to macrophage cell surface patternrecognition receptors or Fc receptors triggers the engulfment ofthe pathogen into specialized intracellular compartments termedphagosomes. Newly formed phagosomes possess no killing ability.This characteristic is acquired through a complex and dynamicphagosome maturation process that results in a highly acidic(<pH 5) proteolytic environment [1]. During maturation, the pro-tein composition of the phagosome is continuously remodeledthrough fusion with early and late endosomes and finally lyso-somes. The phagosome proteome contains well over 100 proteinsthat are involved in phagosome movement along cytoskeletalstructures, phagosome acidification and proteolysis [2].

Ubiquitin has been localized to maturing macrophage phago-somes where it is required for the formation of acidic multivesicu-lar bodies [3], but is not required for phagocytosis or phagosomematuration [4]. Ubiquitination is a posttranslational mechanism

Inc.

ated Molecule; BMDM, boneLPS, lipopolysaccharide; NK,

n fluorescence intensity.f Pathology, Edward Doisyedicine, 1100 South Grand977 6910.

a novel role for ubiquitin in regulating and facilitating bacterialkilling is emerging. Burkholder et al. showed that deubiquitinaseinhibition significantly increases the translocation of inducible ni-tric oxide synthase (iNOS) to the phagosome and enhances the kill-ing of Listeria monocytogenes [6]. Alonso et al. demonstrated thatubiquitin was localized to LAMP1-positive vesicles and cathepsindegradation products of ubiquitin have bactericidal activity againstMycobacterium tuberculosis [7]. Additionally, in vitro studies haveshown that the C-terminal fragment of ubiquitin disrupts themembrane of Mycobacterium smegatis and has antifungal proper-ties [8,9]. Precisely how ubiquitin is trafficked into the lysosomalcompartment is under investigation, but it has been suggested thatubiquitinated protein cargo in autophagosomes is a likely source oflysosomal ubiquitin [10].

Natural Killer Lytic-Associated Molecule is an E3 ubiquitin li-gase and a member of the RING in between RING (RBR) family ofproteins [11]. The N-terminus contains three cysteine-rich do-mains that comprise the RBR structure and ubiquitin ligase activity[12]. NKLAM also has two predicted transmembrane domains [13].In resting cells, NKLAM is very weakly expressed; however treat-ment with cytokines or interleukins such as interferon beta (IFNb)and IL-2 significantly upregulates NKLAM expression [13]. NKLAMis expressed in mononuclear cells such as monocytes and naturalkiller (NK) cells and has been colocalized with granzyme B in thecytolytic granules of NK cells [13]. Such a specific subcellular local-ization implicates NKLAM in the regulation of cytolytic functions

D.W. Lawrence, J. Kornbluth / Cellular Immunology 279 (2012) 46–52 47

and indeed NK cells from mice lacking NKLAM are significantlydefective in lysing tumor target cells [14,15].

In this report, we provide evidence that NKLAM expression isregulated by Escherichia coli lipopolysaccharide (LPS), a Toll-likereceptor 4 (TLR4) agonist. We used IgG-opsonized magnetic latexbeads to show for the first time that NKLAM is a component ofthe macrophage phagosome and translocates to the phagosomeearly in the maturation process. Studies with NKLAM-deficientbone marrow-derived macrophages (BMDM) demonstrate thatNKLAM expression in the phagosome coincides with elevatedlevels of ubiquitinated phagosome proteins. Importantly, we dem-onstrate that both BMDM and peritoneal macrophages lackingNKLAM have a defective killing response against E. coli.

2. Materials and methods

2.1. Bacterial strains and macrophage culture

All experiments on mice were approved by the Animal Care andUse Committees at Saint Louis University and the St. Louis VA. Wildtype C57BL/6 (WT) and corresponding age-matched NKLAM-defi-cient knockout (KO) mice were used in all studies. For isolationof bone marrow, femurs and tibias were flushed with DMEM. Thecollected marrow was resuspended in BM20 media (DMEM sup-plemented with 20% fetal bovine serum, 20% L929-cell conditionedmedia, 2 mM L-glutamine, 100 U/mL penicillin, 100 U/mL strepto-mycin, and 1 mM sodium pyruvate). The bone marrow cells werecultured for 7 days in 100 mm non-tissue culture petri dishes witha partial media change on day 3. To isolate peritoneal macro-phages, ice-cold PBS plus 3% fetal bovine serum (10 mL) was in-jected into the peritoneal cavity of euthanized mice. The fluidwas aspirated from the peritoneal cavity and the cells were resus-pended in DMEM. E. coli (strain JM109) were grown in Luria–Ber-tani (LB) broth overnight at 37 �C with shaking. RAW264.7 andJ774A.1 macrophages were grown in DMEM supplemented with10% fetal bovine serum, 2 mM L-glutamine, 100 U/mL penicillin,100 U/mL streptomycin and cultured at 37 �C in 5% CO2.

2.2. Macrophage stimulation

For experiments using stimulated cells, adherent macrophages(RAW264.7, J774A.1 and bone marrow-derived) were incubatedwith LPS (400 ng/mL) or LPS plus IFNc (100 U/mL) (LPS/IFNc) forat least 18 h at 37 �C or for times indicated. For experiments usingE. coli, macrophage cultures were infected at a multiplicity of infec-tion (MOI) of 10. At the desired time, the cultures were washedbriefly in ice-cold PBS, and then suspended in lysis buffer(65 mM Tris, 0.5% Triton X-100, 137 mM NaCl, 10% glycerol,25 mM sodium orthovanadate, 50 mM sodium fluoride, 10 mM so-dium pyrophosphate, 1 mM EDTA). Protein concentrations weredetermined using bicinchoninic acid (BCA) protein assay reagents(Pierce, Rockford, IL).

2.3. Phagocytosis analysis by flow cytometry

Cultured WT or NKLAM-deficient BMDM (2 � 105) were sus-pended in DMEM plus 20 mM HEPES and kept on ice. Heat-killed,fluorescently-labeled (FITC) E. coli were added to a final MOI of 20and the tubes were incubated at 37 �C for the times indicated. Thecells were then fixed in 2% paraformaldehyde for 10 min on ice.Non-ingested extracellular bacteria were quenched by the additionof 1 mg/mL trypan blue. Macrophage-associated fluorescence wasassessed by flow cytometry. To measure changes in phagosomalpH, E. coli labeled with the pH-sensitive dye pHrodo (Life Technol-ogies, Grand Island, NY) were used as targets and the experiments

were carried out as described above. Flow cytometric data wereanalyzed with FlowJo (Treestar, Ashland, OR).

2.4. Macrophage bacteria killing assay

Wild type and NKLAM-deficient BMDM (3 � 105) were sus-pended in DMEM plus 20 mM HEPES; pH 7.4 and infected withE. coli at an MOI of 10. The cultures were rotated for 20 min at37 �C then washed three times in sterile PBS to remove extracellu-lar bacteria. The infected macrophages were then suspended inDMEM plus 20 mM HEPES and incubated at 37 �C for 90 min. Atthe desired time an aliquot of macrophages was pelleted and lysedin sterile water to release the ingested bacteria. The lysates wereserially diluted and plated on LB agar plates. After overnight incu-bated at 37 �C, the colonies were counted to determine colonyforming units/mL.

2.5. Bead coating protocol

One micron magnetic beads were obtained from Spherotech,Inc. (Lake Forest, IL). Human IgG was added at a saturating concen-tration and the beads were incubated at room temperature for60 min. The coated beads were washed twice in 0.1% BSA/PBS toback-coat the beads and then resuspended at the desired concen-tration in DMEM plus 20 mM HEPES; pH 7.4.

2.6. Phagosome isolation

Macrophages were incubated with IgG-coated magnetic beadsfor 10 min on ice to allow bead binding without ingestion. To in-duce phagocytosis, macrophage suspensions were incubated at37 �C for times indicated. The cell suspensions were then washedtwice with ice-cold PBS to stop ingestion. The cells were resus-pended in 1 mL homogenization buffer (250 mM sucrose, 20 mMHEPES, 0.5 mM EGTA, 0.1% gelatin, pH 7.0) [7] and disrupted with40 strokes of a Wheaton dounce homogenizer. The cellular homog-enate was centrifuged for 5 min at 150g to remove intact cells andnuclei. The post-nuclear supernate was transferred to a new tubeand phagosomes were isolated with a magnet. The phagosome pel-let was washed once in PBS and then lysed in ice-cold lysis buffer.The protein concentration was determined using BCA protein assayreagents and the lysates were solubilized with the addition of 5XSDS–PAGE sample buffer.

2.7. Immunoblotting

Proteins in lysates were separated using SDS–PAGE then trans-ferred to PVDF membrane. Membranes were incubated with pri-mary antibody overnight at 4 �C. The antibodies for LAMP-1 (CellSignaling Technology, Danvers, MA), ubiquitin, and EEA1 (SantaCruz Biotechnology, Santa Cruz, CA), were used at 1:1000. Anti b-actin (Sigma Aldrich, St. Louis, MO) was used at 1:4000 and mono-clonal anti-NKLAM [11] was used at 1:200. After three washes inTBS-T, the blots were probed with HRP-conjugated secondary anti-bodies and the proteins were visualized with BioRad Immun-StarWestern C chemiluminescence kit. Images were captured and ana-lyzed using a BioRad Chemidoc XRS+ imager (BioRad, Inc, Hercules,CA).

2.8. Confocal microscopy

Wild type BMDM were grown on 18 mm acid-cleaned cover-slips until confluent. Heat-killed E. coli labeled with tetramethyl-rhodamine (Sigma Aldrich, St. Louis, MO) were added to themonolayers at an MOI of 10 and the cultures were incubated for60 min at 37 �C. The monolayers were washed with PBS and fixed

48 D.W. Lawrence, J. Kornbluth / Cellular Immunology 279 (2012) 46–52

in 2% paraformaldehyde. The coverslips were blocked and perme-abilized with 5% normal goat serum plus 0.1% saponin for 30 minat room temperature. The coverslips were stained with monoclonalanti-NKLAM at a dilution of 1:50 overnight at 4 �C. After washing inPBS, FITC-labeled goat anti–mouse IgG (1:400) was incubated withthe monolayers for 60 min at room temperature. The coverslipswere washed in PBS then mounted in polyvinyl alcohol mountingmedia. Fluorescent photomicrographs were captured using anOlympus FV-1000 MPE. The z-section images were processed usingthe Colocalization plugin (Pierre Bourdoncle, Institut JacquesMonod) for NIH ImageJ v 1.64.

2.9. Statistical analysis

A two-tailed, unpaired Student’s t-test was used to compare themeans of two groups. A p value of 0.05 or lower was consideredstatistically significant.

3. Results

3.1. NKLAM protein expression is induced by TLR4 agonists

We have previously demonstrated that stimulation of periphe-ral blood monocytes with IFNb strongly enhances NKLAM expres-sion [13]. To further study the potential role of NKLAM inmacrophage function, we investigated whether treatment withTLR4 agonists would affect NKLAM expression. In Fig. 1A,RAW264.7 or J774A.1 macrophages were treated with 400 ng/mLLPS for 0–36 h and NKLAM expression was assessed by Westernblot. Expression of NKLAM was evident by 4 h in both cell types,and was maximal at 16 h. By 24 h of stimulation, NKLAM expres-sion decreased. The anti-NKLAM monoclonal antibody used wasgenerated in our laboratory [13] and recognizes NKLAM as a dou-blet. In Fig. 1B, macrophages were stimulated with a combinationof LPS (400 ng/mL) plus IFNc (100 U/mL) (LPS/IFNc). The overallexpression pattern was similar to cells treated with LPS alone;

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Fig. 1. TLR4 stimulation induces NKLAM expression in macrophages. (A–D)Macrophage cell lines RAW264.7 (top panels) and J774A.1 (bottom panels) wereincubated with LPS (400 ng/mL) (A), LPS (400 ng/mL) plus IFNc (100 U/mL) (B), orE. coli, strain JM109 (C, D). Whole cell lysates were probed for NKLAM expression byWestern blot. (E) NKLAM-deficient (KO) and WT BMDM were treated with LPS(400 ng/mL) for times indicated. Cell lysates were probed for NKLAM expression byWestern blot. b-actin was run as a loading control.

however, the expression of NKLAM was prolonged and more ro-bust. We also used heat-killed E. coli (Fig. 1C) and found an NKLAMexpression pattern similar to that of LPS. Different E. coli: macro-phage ratios were used in Fig. 1D to examine dose dependent ef-fects on NKLAM expression. As the number of E. coli permacrophage was increased, the expression of NKLAM also in-creased. In Fig. 1E, NKLAM-deficient (KO) and WT BMDM weretreated with LPS. In WT BMDM, NKLAM expression was maximalat 5 h and had decreased by 24 h (Fig. 1E). There was no NKLAMprotein observed in NKLAM-deficient macrophages.

3.2. NKLAM is a macrophage phagosomal protein

NKLAM is found in the cytotoxic granules of NK cells and is crit-ical for maximal NK anti-tumor activity [13]. To determinewhether NKLAM was also involved in macrophage-mediated kill-ing, we investigated NKLAM expression in the macrophage phago-some. In Fig. 2A, adherent WT BMDM were incubated with redfluorescent E. coli for 60 min (left panel), fixed and then stainedfor NKLAM. NKLAM had a punctuate, non-nuclear staining pattern(Fig. 2A, middle panel). To determine if NKLAM was localized to thephagosome membrane, we merged the confocal images and se-lected a z-section midway between the top and bottom surfacesof the cell to ensure we were viewing E. coli that were internalizedinto phagosomes. We used a colocalization plugin for ImageJ to de-fine areas of specific colocalization (Fig. 2A, right panel). Theseareas are depicted in white (inset; arrows). We observed signifi-cant levels of NKLAM and E. coli colocalization to the phagosomeperiphery. Biochemical analysis of isolated macrophage phago-somes confirmed the presence of NKLAM in macrophage phago-somes. Untreated and 18 h LPS/IFNc-stimulated RAW264.7macrophages were incubated with IgG-opsonized magnetic beads(Fig. 2B). Treatment with LPS/IFNc stimulated a significant increasein phagosome-localized NKLAM. Similar experiments were alsoperformed using LPS/IFNc-stimulated WT and NKLAM-deficientBMDM (Fig. 2C). NKLAM was present in the phagosomes of WTbut not NKLAM-deficient macrophages. The immunoblots (Fig. 2Band C) were stripped and reprobed for phagosome proteins LAMP1and EEA1 to confirm successful phagosome isolation and to dem-onstrate that the marked changes in phagosomal NKLAM expres-sion were not due to differences in the amount of protein loadedper well. These results demonstrate that NKLAM is a componentof the macrophage phagosome, and is likely localized to the phag-osome membrane.

3.3. NKLAM expression in the phagosome during maturation

We next examined the levels of NKLAM in the phagosome dur-ing the maturation process. In Fig. 3, LPS/IFNc-stimulated WT orNKLAM–KO BMDM were incubated with IgG-opsonized magneticbeads for 10 min on ice to allow bead binding. Cell suspensionswere then switched to 37 �C to initiate phagocytosis. Phagosomeswere isolated at various times from 15–105 min post-ingestion.NKLAM was transiently expressed in the phagosome, and was ob-served as early as 15 min post-ingestion. The levels of NKLAMwithin the phagosome were maximal at 35 min post-ingestionand remained present as late as 105 min post-ingestion. Phago-somes from NKLAM-deficient BMDM did not contain NKLAM atany time point tested. As a control for the kinetics of phagosomematuration, membranes were reprobed for the acid hydrolasecathepsin D. The procathepsin D (pro-CathD) form has been ob-served in macrophage phagosomes as early as 7 min post-ingestion[16]. As the pH of the phagosome decreases, procathepsin D is pro-teolytically cleaved to a 48 kDa intermediate form, then cleavedagain to a 34 kDa mature form [17]. The expression level of thismature form increases in the phagosome over time [16]. As shown

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Fig. 2. NKLAM is localized to the macrophage phagosome. (A) Adherent wild type BMDM were incubated with fluorescent red E. coli (left panel) for 60 min at 37 �C. Themonolayers were fixed in paraformaldehyde and labeled with anti-NKLAM (green, middle panel). Confocal photomicrographs taken at the midpoint of the z-series wereoverlaid (right panel) and processed with the ImageJ Colocalization plugin. Arrows (inset) depict areas of specific colocalization, which are denoted in white. (B) RAW264.7were untreated (�) or stimulated (+) with LPS/IFNc for 18 h at 37 �C. The cells were then incubated with IgG-opsonized magnetic latex beads for 45 min. The phagosomeswere isolated as described in the Section 2. Equal amounts of phagosome protein were immunoblotted for NKLAM and phagosome markers LAMP1 and EEA1. (C) LPS/IFNc-stimulated WT and NKLAM-deficient (KO) BMDM were used in experiments that were identical to (B).

NKLAM KO BMDMWT BMDM15 35 75 105

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Fig. 3. Phagosomal NKLAM expression decreases during maturation. WT orNKLAM–KO BMDM were stimulated with LPS/IFNc for 18 h. The cells wereincubated with IgG-opsonized magnetic beads at 4 �C for 10 min to synchronizebead binding. Ingestion was initiated by incubating the cells at 37 �C. Afterincubation for the times indicated, the phagosomes were isolated as described inSection 2. Phagosome lysates were normalized by protein and immunoblotted forNKLAM and phagosome marker cathepsin D.

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Fig. 4. Ubiquitination of phagosome proteins during maturation. (A) WT andNKLAM–KO BMDM were stimulated with LPS/IFNc for 18 h. The cells were thenincubated with IgG-opsonized magnetic beads at 4 �C for 10 min. After incubationat 37 �C for the times indicated, the phagosomes were isolated and the phagosomeproteins were immunoblotted for ubiquitin. (B) Densitometry was performed oneach lane. Graph represents the ratio of each lane relative to the 15 min time pointfor WT (closed diamonds) and NKLAM–KO (open squares). The immunoblot andgraph are representative of five independent experiments.

D.W. Lawrence, J. Kornbluth / Cellular Immunology 279 (2012) 46–52 49

in Fig. 3 (middle panel), the phagosomes of both WT and NKLAM–KO macrophages contained the 48 kDa intermediate form ofcathepsin D at all time points. Additionally, the mature 34 kDaform of cathepsin D was present in the phagosomes of both celltypes at 15 min and increased during phagosome maturation.These results demonstrate that NKLAM is translocated to the phag-osome early in the maturation process.

3.4. Examination of ubiquitinated phagosomal proteins duringphagosome maturation

Our data demonstrate that NKLAM, a transmembrane E3 ubiq-uitin ligase, is localized to the macrophage phagosome. Previousreports have confirmed the presence of mono- and polyubiquiti-nated proteins in the phagosome membrane [3]. However, studiesdemonstrating that transmembrane ubiquitin ligases are compo-nents of the phagosome and contribute to the ubiquitination ofphagosomal proteins are lacking. Our next set of experimentswas designed to examine the profile of ubiquitinated phagosomal

proteins during phagosome maturation. In Fig. 4, LPS/IFNc-stimulated WT and NKLAM-deficient BMDM were incubated with

50 D.W. Lawrence, J. Kornbluth / Cellular Immunology 279 (2012) 46–52

IgG-opsonized latex beads. Phagosomes were isolated at varioustimes (15–105 min post-ingestion) and the lysates were immuno-blotted with an anti-ubiquitin antibody. A representative blot isshown in Fig. 4A. The phagosomes of both cell types contained sig-nificant amounts of ubiquitinated protein. Interestingly, we ob-served a sharp increase in the amount of ubiquitinated proteinbetween 15 and 35 min post-ingestion in WT phagosomes, whichwas not observed in the phagosomes isolated from NKLAM-deficient macrophages. In both cell types, the levels of ubiquitinat-ed proteins increased during phagosome maturation. In Fig. 4B, thedensitometry values for each lane were determined and were di-vided by the density value of the 15 min time point. This provideda measure of the change in the levels of ubiquitinated proteins foreach time point relative to the earliest time point (15 min) ofmaturation. The levels of ubiquitinated proteins in WT phago-somes increase significantly during a time point (35 min post-ingestion) when NKLAM is maximally localized to the phagosome(Fig. 3, top panel). This increase was not observed in phagosomesfrom NKLAM-deficient macrophages.

3.5. NKLAM expression does not affect uptake of E. coli or phagosomeacidification

We next determined whether a deficiency in NKLAM proteinwould influence the kinetics and/or progression of phagocytosis

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Fig. 5. NKLAM does not affect E. coli ingestion or phagosome acidification. (A, B)Wild type (closed diamonds) and NKLAM-deficient BMDM (open squares) wereincubated with FITC-labeled E. coli for the times indicated, followed by fixation in1% formalin and analysis by flow cytometry. (A) Graph represents the percentage ofmacrophages that were positive for fluorescence. (B) Graph represents the meanfluorescence intensity (MFI) of the macrophage populations. Data represent threeexperiments (± standard deviation). (C–D) Experiments were equivalent to A and Bexcept E. coli were labeled with pH sensitive dye pHrodo to assess changes inphagosomal pH. Data represent six experiments (± standard deviation).

and phagosome acidification. WT or NKLAM-deficient BMDM wereincubated with heat-killed fluorescently-labeled E. coli for up to120 min. At various time points the macrophages were assessedfor bacterial uptake by flow cytometry. As shown in Fig. 5A, WTand NKLAM-deficient BMDM ingested fluorescent E. coli with sim-ilar kinetics. Ingestion increased rapidly within the first 30 min andthen remained constant out to 120 min. The percentage of macro-phages (�70%) that ingested E. coli was also equivalent betweenboth cell types. These results suggest that NKLAM does not influ-ence bacteria ingestion. We also determined the mean fluores-cence intensity (MFI) for each population as a measure of thequantity of E. coli that was ingested per macrophage. As shownin Fig. 5B, there was no significant difference between WT andNKLAM–KO macrophages with respect to the overall number ofbacteria ingested per macrophage.

We used E. coli labeled with a pH sensitive dye (pHrodo) todetermine whether NKLAM is involved in regulating the decreasein phagosomal pH observed during maturation. Wild type orNKLAM-deficient BMDM were incubated with pHrodo-labeledE. coli and the percentage of fluorescently positive macrophageswas assessed by flow cytometry. This population represents thepercentage of macrophages that contained acidified phagosomesfollowing bacterial ingestion. As shown in Fig. 5C, the responsesof both cell types were similar, suggesting that macrophages ofboth genotypes have a similar ability to acidify bacteria-containingphagosomes. Additionally, we determined the MFI for these popu-lations. In this assay, the mean fluorescence intensity can be usedas a measure of the degree of macrophage phagosome acidificat-ion. As shown in Fig. 5D, we found no significant differences be-tween WT and NKLAM-deficient macrophages with respect tothe degree of phagosome acidification. Collectively, these resultssuggest that NKLAM does not play a significant role in either inges-tion of bacteria or regulation of phagosome acidification.

3.6. NKLAM-deficient macrophages are defective in killing E. coli

We have shown previously that NKLAM-deficient NK cells aresignificantly defective in lysing tumor target cells [13,14]. We nextsought to determine whether NKLAM was involved in macrophagebactericidal activity. Bone marrow-derived or peritoneal macro-phages were infected with E. coli (strain JM109) at an MOI of 10.After 90 min, macrophages were lysed in sterile water and viablebacteria were enumerated on LB agar plates. As shown in Fig. 6A,NKLAM-deficient BMDM are significantly defective in killingE. coli. At 90 min, WT BMDM killed an average of 77% of theE. coli while NKLAM–KO BMDM killed 57%. To confirm the ob-served defective killing phenotype in resident macrophages, weisolated peritoneal macrophages from WT and NKLAM-deficientmice and used them as effector cells. As shown in Fig. 6B, WT per-itoneal macrophages killed an average of 40% of the E. coli andNKLAM-deficient macrophages killed 20%. Collectively, these datademonstrate that NKLAM is a positive regulator of the macrophagekilling response against E. coli.

4. Discussion

In the present report, we demonstrate that the E3 ubiquitin li-gase NKLAM is a component of the macrophage phagosome prote-ome. Additionally, our data indicate that NKLAM is a positivemodulator of the macrophage killing response against E. coli andfunctions downstream of bacterial ingestion and phagosomeacidification.

NKLAM was originally isolated from and characterized as an NKcell protein that was regulated by cytokines and involved in the NKcell killing response against tumor cell targets [13,14]. However,

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Fig. 6. NKLAM-deficient macrophages have attenuated bactericidal activity (A) WT(black bar) and NKLAM-deficient (gray bar) BMDM were incubated with live E. coliat an MOI of 10. The cultures were lysed with sterile water after 90 min and viablebacteria were enumerated by colony formation on LB agar plates (⁄, p = 0.03; ±standard deviation; n = 5). (B) Peritoneal macrophages were isolated from wild type(WT, n = 11) and NKLAM-deficient (NKLAM–KO, n = 12) mice and were assessed forE. coli killing ability as in (A). (⁄, p < 0.005).

D.W. Lawrence, J. Kornbluth / Cellular Immunology 279 (2012) 46–52 51

cells of the monocyte/macrophage lineage also express NKLAM.Therefore, we tested the effect of LPS, a TLR4 agonist, on NKLAMexpression in macrophages. Treatment of RAW264.7 and J774A.1macrophages with LPS induced a strong and transient increase inNKLAM expression. J774A.1 cells appear significantly less respon-sive than RAW 264.7 cells to LPS stimulation with respect toNKLAM protein expression. In support of this observation, a studyfound that RAW264.7 macrophages produced more TNFa in re-sponse to LPS than J774A.1 [18]. Wild type BMDM macrophageswere the most responsive to LPS and demonstrated elevatedNKLAM levels at 5 h. In all cases, NKLAM expression decreases after24 h. This expression profile suggests that macrophage NKLAMprotein is not preexisting but is rapidly translated upon stimula-tion. A related RBR ubiquitin ligase, Parkin, is also regulated byLPS [19]. Mutations in the parkin gene (PARK2) are associated withearly-onset parkinsonism and recessive juvenile parkinsonism[20,21]. Interestingly, polymorphisms in PARK2 have been associ-ated with increased susceptibility to infection by Salmonella typhi,Salmonella paratyphi, and Mycobacterium leprae [22–24]. Thus, thecellular functions of RBR ubiquitin ligases are diverse and may beinvolved in cell signaling pathways that are common to neurolog-ical and infectious diseases.

Depending on the method of detection, the number of proteinsassociated with the phagosome range from over 100 [2] toseveral thousand [25]. These proteins are involved in phagosome

trafficking, protein degradation, phagosome acidification, and anti-gen presentation [26]. We found that membrane-associatedNKLAM was a component of stimulated macrophage phagosomes.Previous research has shown that ubiquitin is translocated to thematuring phagosome where it contributes to the formation ofmultivesicular bodies [3,4]. However, studies that examine the po-tential contribution of ubiquitin ligases to the ubiquitination ofphagosome proteins are lacking. After ingestion of opsonizedbeads, NKLAM expression in the phagosome was maximal at35 min, suggesting that NKLAM may be translocated to the phago-some via the endosomal pathway. The levels of NKLAM in thephagosome decreased over time in WT BMDM. Whether NKLAMis removed from the phagosome or degraded is currently underinvestigation. Loss of phagosome proteins can occur through fis-sion of vesicles from the phagosome membrane or through theinvagination of the phagosome limiting membrane during theformation of multivesicular bodies, which are then degradedafter fusion with lysosomes [27]. NKLAM is also capable of self-ubiquitination, which may target it for degradation by protea-somes at the phagosome surface.

Our studies indicate that NKLAM contributes to the ubiquitina-tion of phagosomal proteins. However, the presence of ubiquitinat-ed proteins in the phagosomes of NKLAM-deficient macrophagessuggests that additional ubiquitin ligases are associated with mac-rophage phagosomes.

There are few studies that examine the role of ubiquitin inphagocytosis. Booth et al. demonstrated that ubiquitin conjugationmachinery was not required for phagocytosis of opsonized sheepred blood cells [4]. Our studies with NKLAM-deficient mice supportthis observation, in that we found no difference between WT andNKLAM-deficient macrophages in the rate of ingestion or the num-ber of E. coli ingested per macrophage. In contrast, Silva et al. dem-onstrated that a protein named pallbearer, acting as an E3 ligase,was required for efficient phagocytosis of apoptotic cells by Dro-sophila macrophages [28]. Thus, the involvement of ubiquitinationin regulating phagocytosis may vary depending upon the type ofhost macrophage and the target being ingested.

We show that both bone marrow-derived and peritoneal mac-rophages lacking NKLAM are significantly defective in mountingan effective killing response against E. coli. At present, preciselyhow ubiquitin ligases function in macrophage bactericidal activityis unknown. Ubiquitination regulates membrane protein sorting[29]; thus one could envision a role for NKLAM in regulating thetrafficking of proteins related to bactericidal functions. In supportof this concept, recent studies have demonstrated that inhibitionof cellular deubiquitinases increased the trafficking of iNOS tophagosomes and significantly augmented macrophage L. monocyt-ogenes killing [6]. Recent studies have also suggested that the ubiq-uitination machinery normally associated with autophagy isinvolved in recognizing and degrading bacteria that escape thephagosome into the cytoplasm. In this process, cytoplasmic bacte-ria become polyubiquitinated, and become associated with auto-phagosomes [30]. Autophagosomes then fuse with lysosomes andthe bacteria cargo are degraded. In this scenario, bacterial ubiqui-tination would be a critical step towards efficient removal of cyto-plasmic pathogens. Further studies are needed to conclusivelydetermine which ubiquitin ligases are involved in this processand which bacterial proteins are targets for ubiquitination.

5. Conclusion

We have demonstrated that RBR family E3 ubiquitin ligaseNKLAM is a novel macrophage phagosome protein. Our studiessuggest that NKLAM functions downstream of the initial phago-cytic event to positively regulate macrophage bactericidal activity.

52 D.W. Lawrence, J. Kornbluth / Cellular Immunology 279 (2012) 46–52

The elucidation of potential NKLAM substrates will be critical fordetermining the mechanism of action of this ubiquitin ligase. Weanticipate that future studies will solidify the role of NKLAM, andpossibly other RBR family ligases, as a key component of the leuko-cyte bactericidal machinery. The role of ubiquitination as part ofthe leukocyte killing response provides additional therapeutic tar-gets for modulating bacterial killing during infection.

Acknowledgments

We would like to thank Gail Gullickson for animal care, and ex-pert technical assistance. We also thank Robin Chamberland formany insightful discussions.

These studies were supported in part by NIH GrantR56AI089758 and by a Grant (1 IO1BX000705) from the Depart-ment of Veterans Affairs, Veterans Health Administration, Officeof Research and Development.

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