IgM-Dependent in Microglia Is Mediated by 3, Not Fc α/µ Receptor

This information is current as Jonathan R. Weinstein, Yi Quan, Josiah F. Hanson, Lucrezia of September 25, 2021. Colonna, Michael Iorga, Shin-ichiro Honda, Kazuko Shibuya, Akira Shibuya, Keith B. Elkon and Thomas Möller J Immunol 2015; 195:5309-5317; Prepublished online 23 October 2015;

doi: 10.4049/jimmunol.1401195 Downloaded from http://www.jimmunol.org/content/195/11/5309

Supplementary http://www.jimmunol.org/content/suppl/2015/10/23/jimmunol.140119 Material 5.DCSupplemental http://www.jimmunol.org/ References This article cites 56 articles, 9 of which you can access for free at: http://www.jimmunol.org/content/195/11/5309.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

IgM-Dependent Phagocytosis in Microglia Is Mediated by Complement Receptor 3, Not Fca/m Receptor

Jonathan R. Weinstein,*,1 Yi Quan,*,1 Josiah F. Hanson,* Lucrezia Colonna,† Michael Iorga,* Shin-ichiro Honda,‡ Kazuko Shibuya,‡ Akira Shibuya,‡ Keith B. Elkon,† and Thomas Mo¨ller*

Microglia play an important role in receptor-mediated phagocytosis in the CNS. In brain abscess and other CNS infections, invading bacteria undergo opsonization with Igs or complement. Microglia recognize these opsonized pathogens by Fc or complement re- ceptors triggering phagocytosis. In this study, we investigated the role of Fca/mR, the less-studied receptor for IgM and IgA, in microglial phagocytosis. We showed that primary microglia, as well as N9 microglial cells, express Fca/mR. We also showed that anti-Staphylococcus aureus IgM markedly increased the rate of microglial S. aureus phagocytosis. To unequivocally test the role of Fca/mR in IgM-mediated phagocytosis, we performed experiments in microglia from Fca/mR2/2 mice. Surprisingly, we found Downloaded from that IgM-dependent phagocytosis of S. aureus was similar in microglia derived from wild-type or Fca/mR2/2 mice. We hypoth- esized that IgM-dependent activation of complement receptors might contribute to the IgM-mediated increase in phagocytosis. To test this, we used immunologic and genetic inactivation of complement receptor 3 components (CD11b and CD18) as well as C3. IgM-, but not IgG-mediated phagocytosis of S. aureus was reduced in wild-type microglia and macrophages following preincu- bation with an anti-CD11b blocking Ab. IgM-dependent phagocytosis of S. aureus was also reduced in microglia derived from CD182/2 and C32/2 mice. Taken together, our findings implicate complement receptor 3 and C3, but not Fca/mR, in IgM- http://www.jimmunol.org/ mediated phagocytosis of S. aureus by microglia. The Journal of Immunology, 2015, 195: 5309–5317.

hagocytosis is a multistep and receptor-mediated process. expressed on B cells and macrophages, where it has been shown to It is initiated by particle recognition and can be separated mediate uptake of IgM-Ag immune complexes (8, 9). In addition to P experimentally into two steps, as follows: 1) particle at- opsonization by Abs, phagocytic objects (microorganisms or cells) tachment to the cell surface and 2) particle ingestion by cells (1). can be opsonized by complement and recognized by complement Phagocytosis is mediated by a wide variety of cell surface receptors receptors, including , complement receptor 3

that bind directly or indirectly, through , to particles (2). Fc (CR3), complement receptor 4, and C1qR(P) (2, 11). by guest on September 25, 2021 receptors, specifically the Fcg receptor subtypes that recognize IgG, Microglia, the resident tissue macrophages of the CNS, are active are well studied for their role in phagocytosis (3, 4). However, other sensors and versatile effector cells in the normal and pathologic brain Igs, including IgM (5, 6) and IgA (7), are also capable of opsonizing (12). Microglia shift activity states depending on the surrounding pathogens and playing a role in phagocytosis. A recently discovered microenvironment. Under normal conditions, they are characterized phagocytosis-related Fc receptor known as Fca/mR (CD351) rec- by a small cell body with fine, ramified processes and low expres- ognizes IgM and IgA, but not IgG (8–10). It is a type 1 trans- sion of surface Ags. In response to brain injury, ischemia, and in- membrane with an extracellular Ig-like domain. Fca/mRis flammatory stimuli, microglia rapidly transform into an activated associated with morphological changes, proliferation, migration to the site of injury, elaboration of both neurotoxic and *Department of Neurology, School of Medicine, University of Washington, Seattle, WA neurotrophic factors, as well as increased phagocytosis (12–14). In- 98195; †Division of Rheumatology, Department of Medicine, School of Medicine, University of Washington, Seattle, WA 98195; and ‡Department of Immunology, vading pathogens require opsonization by Ig and complement fixa- Institute of Basic Medical Sciences, Graduate School of Comprehensive Human tion for efficient recognition and phagocytosis by Fc and complement Sciences, University of Tsukuba, Ibaraki 305-8575, Japan receptors in microglia (2, 15). Microglial Fc and complement re- 1J.R.W. and Y.Q. contributed equally to this work. ceptors have been implicated in the pathophysiology of bacterial ORCIDs: 0000-001-6080-9758 (J.R.W.); 0000-0003-2572-8893 (Y.Q.). brain abscesses (16). Fc and complement receptors also represent Received for publication May 9, 2014. Accepted for publication September 29, 2015. potential molecular targets for pharmacologic therapy in multiple This work was supported by American Heart Association Grant-In-Aid 0750078Z (to sclerosis (3), Alzheimer’s (17–19), and Parkinson’s disease (20). T.M.), National Institutes of Health/National Institute of Arthritis and Musculoskel- Although IgG-mediated phagocytosis (via Fcg receptors) in the etal and Skin Diseases Grant F32AR065837 (to L.C.), and National Institutes of Health/National Institute of Neurological Disorders and Stroke Grant NS065008 CNS is well described (3), there is little characterization to date of (to J.R.W.). IgM-mediated phagocytosis in brain. In this study, we investigate Address correspondence and reprint requests to Prof. Jonathan R. Weinstein, Depart- IgM-induced phagocytosis of the bacterial pathogen Staphylo- ment of Neurology, School of Medicine, University of Washington, Box 356465, coccus aureus in microglia and characterize the role of Fca/mRin 1959 NE Pacific Street, Seattle, WA 98195. E-mail address: [email protected]. edu this process. The online version of this article contains supplemental material. Abbreviations used in this article: CR3, complement receptor 3; MSFM, macrophage Materials and Methods serum-free medium; pMG, primary microglia; P/S, penicillin/streptomycin; qRT- Solutions and reagents PCR, quantitative real-time PCR; WT, wild-type. Fluorescein-labeled S. aureus was obtained from Invitrogen. Anti-S. au- Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 reus mAbs (IgM clone 11-248.2 and IgG3 clone 11-232.3) were purchased www.jimmunol.org/cgi/doi/10.4049/jimmunol.1401195 5310 CR3 MEDIATES IgM-DEPENDENT PHAGOCYTOSIS IN MICROGLIA from QED Bioscience (San Diego, CA). Anti-CD11b mAb as well as S. aureus IgM, 2) isotype control IgM, 3) anti-S. aureus IgG, or 4) isotype isotype controls for both anti-S. aureus and anti-CD11b Abs were obtained control IgG, were added to the cells to assess phagocytosis. from BD Biosciences (San Jose, CA). Recombinant mouse GM-CSF, IFN-g, We also used microscopy to confirm phagocytosis in serum-starved and IL-4 were purchased from R&D Systems (Minneapolis, MN). All mouse pMG. After 30-min incubation with each of the four S. aureus/ solutions were freshly prepared from frozen stock solutions or lyophilized Ab reaction mixes, extracellular fluorescence was quenched with trypan preparations. All materials were handled in a sterile manner using blue. Primary microglia were fixed, counterstained with DAPI, and then endotoxin-free microfuge tubes (Eppendorf/Fisher Scientific, Santa Clara, imaged and photographed under both fluorescent and differential inter- CA), polypropylene tubes (BD Labware, Franklin Lakes, NJ), polystyrene ference contrast illumination using a Zeiss Axiovert 200M microscope. culture vessels (BD Labware), serological pipettes (Costar/Corning, Corning, NY), precision pipette tips (Rainin Instruments, Oakland, CA), Quantification of cell surface expression of Fca/m receptor water (Associates of Cape Cod), and PBS (Life Technologies/Invitrogen, Carlsbad, CA). To quantify Fca/mR cell surface protein expression, pMG or N9 cells were cultured on poly-ornithine (Sigma-Aldrich, St. Louis, MO)–coated plates Animals and cell culture in MSFM, and cells were dislodged without trypsin using 2 mM EDTA/ PBS. Cells were fixed for 10 min at room temperature in a phosphate The mouse microglial cell line N9 was a gift of M. Righi (International buffer containing 2% paraformaldehyde/0.6% methanol (Fisher Scientific, School for Advanced Studies, Trieste, Italy) and was cultured in accordance Kent, WA), 2.5 mM sodium periodate (Fisher), and 15 mM L-lysine with the original publication (21). In brief, cells were cultured in high- (Sigma-Aldrich), and then blocked with 1 mg/ml ice-cold mouse sera (BD glucose DMEM (Life Technologies/Invitrogen), supplemented with 10% Biosciences) plus 50 mg/ml unlabeled streptavidin (BD Biosciences) in heat-inactivated FBS (HyClone, Logan, UT), and penicillin/streptomycin PBS. Following a wash step, primary Ab incubations were carried out with (P/S; 50 IU/50 mg/ml; Mediatech/Corning, Manassas, VA). Cells were either 50 mg/ml rat anti-Fca/mR mAb (clone TX-7) (27) or isotype control passaged weekly with 0.05% trypsin (Life Technologies/Invitrogen) and se- (BD Biosciences). For detection, we used biotinylated mouse anti-rat IgG rum starved in macrophage serum-free medium (MSFM; Life Technologies/ 2b (BD Biosciences), followed by 2 mg/ml PE-Cy7-streptavidin (BD Downloaded from Invitrogen) for at least 24 h before each experiment, as detailed below. Biosciences). Staining was quantified on a FACSAria cytometer/sorter (BD 2/2 The Fca/mR mice used for these studies were as previously reported Biosciences) and analyzed with FlowJo software (Tree Star). For our 2/2 2/2 (10). The CD18 (22) and C3 (23) mice were from The Jackson primary cultures, we first selected confirmed pMG by flow cytometry Laboratory (Bar Harbor, ME). All mice were on the same genetic back- based on their expression of CD11b (96% of total cell population). ground (C57BL/6). Primary microglia (pMG) were prepared from the 2 2 2 2 2 2 cortex of newborn (p4) wild-type (WT), Fca/mR / , CD18 / ,orC3 / RNA isolation, reverse , and quantitative mice, as previously described (24, 25). In brief, cortical tissue was care- real-time PCR fully freed from vessels and meninges, digested with 50 ng/ml http://www.jimmunol.org/ DNase, triturated, and washed. Cortical cells were cultured in DMEM/10% To detect the mRNA expression of Fca/mR in mouse microglia, RNA FBS with P/S plus 2 ng/ml GM-CSF (R&D Systems) for 11–50 d isolation (including on-column DNase digestion), reverse transcription, (media change every 3–4 d). Microglia were separated from underlying and quantitative real-time PCR (qRT-PCR) were done, as previously de- astrocytic monolayer by gentle agitation, spun down (100 3 g for 10 min). scribed (28). In brief, qRT-PCR was performed using the 7500 Real Time Cell pellet was resuspended in DMEM/10% FBS with P/S plus 2 ng/ml PCR System (Applied Biosystems). SYBR GreenER qPCR SuperMix GM-CSF and plated on BD Primaria culture dishes and plates (Falcon, Universal Kit (Invitrogen) was used to amplify Fca/mR and endogenous St. Laurent, QC, Canada). Nonadherent cells were removed 30–60 min control HPRT (26). The following components were combined per 40 ml after plating by changing the medium, and adherent microglia were in- reaction volume: .1 mg cDNA, 20 ml SYBR GreenER qPCR SuperMix, cubated for 24 h in culture medium before being serum starved in MSFM 50 nM ROX, 500 nM mouse Fca/mR forward primer 59-CTG CTT CTA plus 0.2 ng/ml GM-CSF for 24 h. ATT GCT GCT CTG-39 and reverse primer 59-GCT TAT CTG GTA GGA AAT GTG TC-39, or 200 nM HPRT primers. Cycling conditions were as by guest on September 25, 2021 Phagocytosis assays follows: 1) 50˚C for 2 min; 2) 95˚C for 10 min; 3) 40 cycles, with each cycle consisting of 95˚C for 15 s and 60˚C for 1 min. Fluorescent data were Ig-induced phagocytosis assays were done, as previously described (26). acquired at the 60˚C step. Melting curve analysis was carried out to verify In brief, after reconstitution, according to manufacturer’s instructions, single-species PCR products. All the experiments had a no-template fluorescein-labeled S. aureus (Molecular Probes/Life Technologies, Grand negative control, and only intron-spanning primers were used. Data were Island, NY) was incubated overnight at 4˚C with equimolar concentrations analyzed using Sequence Detection Software v1.3 (Applied Biosystems), of different anti-S. aureus or control Ab isotypes (20 mg specific anti- as previously described (28). For qRT-PCR quantifying expression of S. aureus IgG/mg bacteria; 20 mg isotype IgG/mg bacteria; 120 mg specific CD11b, CD18, and C3 mRNA transcripts, singleplex amplification was anti-S. aureus IgM/mg bacteria; 120 mg isotype IgM/mg bacteria). Before performed on a StepOnePlus Real-Time PCR System (Applied Bio- use, Ab-opsonized S. aureus was washed with PBS once and then spun systems). Cycling conditions consisted of the following: 1) 95˚C for 10 down and resuspended in MSFM. The various Ab/S. aureus reaction mixes min; 2) 50 cycles of 95˚C for 10 s; and 60˚C for 30 s. Primer and probe were then incubated with serum-starved mouse pMG or N9 cells. After 30 sequences are shown in Supplemental Table I. Relative expression min, extracellular fluorescence was quenched with 0.1% trypan blue (Life was calculated using the DD cycle threshold method in which samples Technologies). Then pMG were dislodged from wells with PBS containing were normalized to the cycle threshold geometric mean of the house- 0.25% trypsin plus 2 mM EDTA (Life Technologies) (5 min at 37˚C), and keeping (HPRT, EIF4a2, and ATP5b). Numerical data are given as intracellular fluorescence was determined and analyzed using a FACScan2 the mean 6 SEM of the normalized mean values from each independent flow cytometer (BD Biosciences, San Jose, CA) and FlowJo software (Tree experiment. All experiments were carried out in triplicate. Star, Ashland, OR). For analysis, we set a threshold for basal (non-Ab– mediated) phagocytosis by processing cells using the isotype control Ab Statistical analysis reaction mixes and setting the lower border of a marker gate to encompass only the 2.5% of total cellular events that had the highest median fluo- Statistical evaluation was carried out using PRISM software (GraphPad, rescent intensity values. This same gate was then applied to results from San Diego, CA). Differences between two groups were analyzed by the reaction mixes containing specific anti-S. aureus IgG or IgM Abs. This Student t test. Multiple comparisons were made using one-way ANOVA allowed us to determine percentage of cells that had undergone specific with Bonferroni posttest. The p value ,0.05 was considered to be significant. Ab-mediated endocytosis (true positives). Data are presented as the ratio of Data are given as mean 6 SEM. true positives in a specific Ab group to that of the matching isotype control Ab group. We will refer to this ratio as fold increase of phagocytosis. In a few instances (Figs. 6B, 8), to facilitate a mechanistic comparison of Results the effects of anti-CD11b Ab on anti-S. aureus IgM- and IgG-mediated Surface expression of the Fca/m receptor in microglia phagocytosis of S. aureus, we normalized the fold increase of phagocy- tosis for each Ig class to that induced by the corresponding anti-S. aureus Uptake of IgM- and IgA-, but not IgG-Ag immune complexes in IgM or IgG alone and presented the data as percentage of control. N9 cells macrophages is mediated by Fca/mR (8). We assessed expression were processed for phagocytosis assay in a similar manner to pMG, except of Fca/mR by flow cytometry and found robust cell surface ex- no trypsin was required for dislodgement. For CD11b blocking experi- ments, pMG were preincubated with 1 mg/ml anti-CD11b IgG or isotype pression in both pMG (Fig. 1A) and N9 cells (Fig. 1B). Expression control IgG for 1 h, and then reaction mixes composed of fluorescein- of Fca/mR in both pMG and N9 cells was confirmed by qRT-PCR labeled S. aureus combined with either of the following, 1) anti- (Fig. 2A). The Journal of Immunology 5311

IgM-induced phagocytosis in microglia To test the function of Fca/mR in mouse pMG, we assessed the phagocytosis of IgM-opsonized S. aureus and found that IgM opsonization increased phagocytosis ∼18-fold over isotype IgM control (Fig. 3A, 3C). In N9 cells, anti-S. aureus IgM induced a 12-fold increase in phagocytosis relative to isotype control IgM (Fig. 3D). Differing affinities/avidities of IgM and IgG for the same Ag in our experimental setup preclude direct comparisons of functional effects induced by different Ig classes. Nevertheless, we considered characterization of both IgM- and IgG-mediated Downloaded from http://www.jimmunol.org/

FIGURE 2. Cytokine-induced regulation of Fca/mR mRNA and IgM- mediated phagocytosis in microglia. (A) Results of qRT-PCR experiment on RNA extracted from mouse pMG or N9 cells that were either unsti- mulated (control) or stimulated with 10 ng/ml IL-4 or 10 U/ml IFN-g for 24 h. Findings are presented as fold increase in steady-state levels of Fca/ mR mRNA in the stimulated groups relative to unstimulated control. **p , B 0.01 versus corresponding unstimulated controls. ( ) Bar graph quantify- by guest on September 25, 2021 ing IL-4– or IFN-g–induced changes in anti-S. aureus IgM- or isotype control IgM-induced fold changes in S. aureus phagocytosis in pMG (see Materials and Methods for detail). Total n = 6 (two wells per experimental group from three separate experiments). **p , 0.01 versus isotype control, ###p , 0.001 versus anti-S. aureus IgM signal in unstimulated control group.

phagocytosis of S. aureus important in this setting to allow us to examine Ig class-specific mechanisms involved. Thus, we used a S. aureus-specific IgG to quantify IgG-mediated phagocytosis. The rate of IgG-opsonized S. aureus increased relative to that of isotype IgG control (Fig. 3B, 3C) by a factor of ∼3-fold (Fig. 3C). In N9 cells, IgG-induced phagocytosis increased 7-fold relative to the corresponding isotype control IgG group (Fig. 3D). To visu- alize internalization of S. aureus, we performed fluorescent mi- croscopy. Mouse pMG exposed to reaction mixes containing either specific anti-S. aureus IgM or IgG qualitatively accumu- lated appreciably greater amounts of S. aureus in their cytoplasms FIGURE 1. Cell surface expression of Fca/mR protein in microglia. compared with pMG exposed to isotype control Ab reaction mixes Flow cytometry histogram plots showing fluorescent intensity signal for (Fig. 4). Absence of noncellular associated S. aureus fluorescence + populations of CD11b pMG (A) or N9 cells (B) that are either unstained confirmed the effectiveness of trypan blue quenching (Fig. 4). (black shaded histograms) or stained with anti-Fca/mR (black line) or These imaging findings are consistent with and complement the isotype control (shaded histogram) Abs. Detection was with biotinylated quantitative flow cytometry results from Fig. 3. mouse anti-rat IgG, followed by PE-Cy7-streptavidin, as described in Materials and Methods. Marker gates in both (A) and (B) indicate per- Cytokine-induced regulation of Fca/mR mRNA and IgM- centage of anti-Fca/mR Ab-stained microglia that had fluorescent intensity induced phagocytosis in microglia readings greater than isotype control background (i.e., signal .97.5% of microglia stained with isotype control Ab). Legend below figure shows We previously demonstrated that –derived cytokines, in- median fluorescent intensity (FI) values for both pMG and N9 cells either cluding IL-4 and IFN-g, differentially regulated expression of Fcg unstained or stained with anti-Fca/mR or isotype control (ISO) Abs. Re- receptor subtypes in microglia (26). These same cytokines mod- sults shown are representative data from three experiments. ulated IgG-mediated phagocytosis in microglia in a manner that 5312 CR3 MEDIATES IgM-DEPENDENT PHAGOCYTOSIS IN MICROGLIA

FIGURE 3. Quantification of Ig-dependent phagocytosis of Staphylococcus aureus (SA) in microglia. Flow cytometric histogram plots show the fol- lowing: (A) tonic/baseline (shaded black), isotype control IgM (shaded gray)- and anti-S. aureus IgM (black line)-dependent phagocytosis in mouse pMG, or (B) tonic/baseline (shaded black), isotype control IgG (shaded gray)- and anti-S. aureus IgG (black line)-dependent phagocytosis in pMG. Marker gates in both (A) and (B) indicate percentage of anti-S. aureus Ab-dependent pMG with FITC fluorescent intensity signal greater than background (see Materials Downloaded from and Methods for detail). (C) Bar graph quantifying anti-S. aureus IgM- or IgG-dependent fold increase in S. aureus phagocytosis (relative to isotype control IgM or IgG, respectively) in pMG (see Materials and Methods for detail). (D) Bar graph quantifying anti-S. aureus IgM- or IgG-dependent fold increase in S. aureus phagocytosis (relative to isotype control IgM or IgG, respectively) in mouse microglia cell line N9. Total n = 6 (two wells per experimental group from three separate experiments). *p , 0.05 versus isotype control Ig group, **p , 0.01 versus isotype control Ig group.

was consistent with the following: 1) their effects on Fcg receptor did not readily explain the effects of these same cytokines on http://www.jimmunol.org/ subtype expression and 2) the known functions of the specific Fcg IgM-mediated phagocytosis of S. aureus in microglia. receptor subtypes that were regulated (26). We sought to deter- a m mine in this work whether a similar pattern would emerge for Effect of genetic of Fc / R on IgM-induced cytokine-induced regulation of Fca/mR and IgM- phagocytosis in microglia mediated phagocytosis of S. aureus in microglia. Treatment with To unequivocally test for the role of Fca/mR in IgM-mediated IL-4 for 24 h did not significantly alter expression of Fca/mR phagocytosis of S. aureus in microglia, we cultured pMG derived steady-state mRNA levels in either pMG or N9 cells. However, from either WT or Fca/mR2/2 mice and characterized IgM-induced treatment with IFN-g did induce a marked decrease in Fca/mR phagocytosis (Fig. 5). Surprisingly, we found no differences in by guest on September 25, 2021 expression in both pMG and N9 cells (Fig. 2A). In contrast to the IgM-induced phagocytosis of S. aureus in WT versus Fca/mR2/2 absence of an effect of IL-4 on Fca/mR expression, treatment with microglia (Fig. 5). Thus, absence of Fca/mR had no effect on IgM- IL-4 for 24 h significantly reduced IgM-mediated phagocytosis of induced phagocytosis of S. aureus in mouse microglia. S. aureus in pMG. Treatment with IFN-g, despite its dramatic lowering of Fca/mR mRNA expression, had no effect on IgM- Role for CR3 in IgM-mediated phagocytosis in microglia mediated phagocytosis (Fig. 2B). Thus, for both IL-4 and IFN-g, Besides its reported role in Fca/mR-mediated phagocytosis, IgM cytokine-induced regulation of Fca/mR occurred in a manner that is also a potent activator of complement (29). To reconcile our

FIGURE 4. Fluorescent microscopy images demonstrating Ig-dependent phagocytosis of Staphylococcus aureus (SA) in microglia. (A) Phagocytosis of isotype control (iso) IgM-opsonized FITC-S. aureus in mouse pMG. (B) Phagocytosis of anti-S. aureus IgM-opsonized FITC-S. aureus in pMG. (C) Phagocytosis of iso IgG-opsonized FITC-S. aureus in pMG. (D) Phagocytosis of anti-S. aureus IgG-opsonized FITC-S. aureus in pMG. Phagocytosis assay with S. aureus opsonized by indicated Ab was carried out, as described in Materials and Methods. Fluorescent and differential interference contrast (DIC) images were acquired to show Ig-induced phagocytosis. Results shown are representative images from three experiments. For each experiment, there were three replicates in each treatment group, and three different fields of view were imaged in each replicate. Original magnification 3400. The Journal of Immunology 5313

receptors, including CR3 (31). Microglia have been previously reported to express and secrete C3 (32). To determine whether C3 plays a role in IgM-mediated phagocytosis of S. aureus in microglia, we compared rates of phagocytosis in microglia derived from WT versus C32/2 mice. We found a significant (∼30%) re- duction in the rate of IgM-mediated phagocytosis in C32/2 microglia (Fig. 8A). In contrast, we found no difference in the rate of IgG-mediated phagocytosis of S. aureus in WT versus C32/2 microglia (Fig. 8B). It is important to note that the microglia used for these studies had been cultured exclusively in heat-inactivated serum prior to serum starvation, thus reducing the potential ex- posure to any exogenous source of complement. It is also im- portant to emphasize that the microglia were thoroughly washed and serum starved prior to the phagocytosis assays; thus, it is unlikely that any potential residual C3 in the heat-inactivated se- rum would be present in sufficient quantities to affect the results. Thus, these data strongly suggest that microglial production of C3 is required for optimal IgM-, but not IgG-mediated phagocytosis

of S. aureus in microglia. Downloaded from

A http://www.jimmunol.org/

FIGURE 5. IgM-induced phagocytosis in WT versus Fca/mR2/2 de- rived microglia. (A) Flow cytometry histogram plots showing fluorescent intensity signal for populations of WT and Fca/mR2/2 mouse pMG that were incubated with FITC-S. aureus opsonized either by anti-S. aureus B IgM (black lines) or isotype control IgM (shaded histograms). Marker gates indicate percentage of anti-S. aureus IgM-induced pMG with FITC by guest on September 25, 2021 fluorescent intensity signal greater than background (see Materials and Methods for detail). (B) Bar graph quantifying anti-S. aureus IgM-induced fold increase in S. aureus phagocytosis (relative to isotype control IgM) in WT or Fca/mR2/2 pMG (see Materials and Methods for detail). n.s. in- dicates no significant difference between WT and Fca/mR2/2. Total n =6 (two wells per experimental group from three separate experiments). observations with current knowledge of microglial phagocytosis, we hypothesized that IgM opsonization of S. aureus leads to IgM- mediated complement activation and subsequent phagocytosis via a complement receptor-mediated mechanism, specifically via CR3. CR3 is a heterodimeric protein composed of CD11b and CD18 (30). CR3 plays a well-documented role in the phagocytic function of microglia in CNS diseases (30). To test our hypothesis, FIGURE 6. Effect of anti-CD11b blocking Ab on anti-S. aureus IgM- or we first examined the effect of anti-mouse CD11b Ab on IgM- anti-S. aureus IgG-mediated phagocytosis in WT microglia. (A) Repre- induced phagocytosis of S. aureus. Preincubation of pMG with sentative flow cytometry histogram plots showing fluorescent intensity anti-CD11b Ab inhibited IgM-induced phagocytosis of S. aureus signal for populations of WT pMG that were preincubated with either anti- by ∼70%, whereas preincubation with an isotype control Ab CD11b blocking Ab or isotype control Ab and then incubated with FITC- resulted in no change in IgM-induced phagocytosis (Fig. 6). In S. aureus opsonized either by anti-S. aureus IgM (black lines) or isotype contrast, preincubation with anti-CD11b blocking Ab did not inhibit control IgM (shaded histograms). Marker gates indicate percentage of anti- IgG-mediated phagocytosis of S. aureus in microglia (Fig. 6B). To S. aureus IgM-induced pMG with FITC fluorescent intensity signal greater B confirm that CR3 plays a role in IgM-mediated phagocytosis of than background (see Materials and Methods for detail). ( ) Bar graph quantifying effects of anti-CD11b blocking Ab or isotype control Ab on S. aureus, we determined whether deletion of the gene encoding anti-S. aureus IgM- or IgG-mediated increase in S. aureus phagocytosis in CD18 altered IgM-mediated phagocytosis in pMG. Genetic deletion pMG (see Materials and Methods for detail). For clarity of comparison, of CD18 attenuated IgM-mediated phagocytosis of S. aureus in data are normalized to values with corresponding anti-S. aureus IgM or pMG by ∼60% relative to WT (Fig. 7). IgG Ab alone, respectively, and presented as percentage of fold increase. Role for C3 in IgM-mediated phagocytosis in microglia n.s. indicates not significantly different compared with anti-S. aureus IgM alone group. Total n = 6; two wells per experimental group from three C3 is a central component of the and promotes separate experiments. ***p , 0.001 compared with anti-S. aureus IgM Ab phagocytosis of pathogens by binding to cellular complement alone, ###p , 0.001 compared with anti-S. aureus IgG Ab alone. 5314 CR3 MEDIATES IgM-DEPENDENT PHAGOCYTOSIS IN MICROGLIA

each other out and resulted in no net change in the IgM-mediated phagocytic function of the cells (Fig. 2B).

Discussion The key findings in this study include the following: 1) microglia express the IgM receptor Fca/mR at their cell surface; 2) genetic deletion of Fca/mR does not alter IgM-mediated phagocytosis of S. aureus in microglia; 3) disruption of another phagocytic re- ceptor, CR3, in microglia (via either Ab blockade or genetic deletion) significantly attenuates IgM-, but not IgG-mediated phagocytosis of S. aureus; and 4) genetic deletion of C3 signifi- cantly reduces IgM-, but not IgG-mediated phagocytosis of S. aureus. Taken together, these findings suggest that biologically relevant IgM-mediated phagocytosis in brain can be carried out by microglia and that this process is dependent on CR3 and, at least in part, on C3 as well, but not on Fca/mR. To our knowledge, this is the first demonstration of Fca/mR expression in microglia. Our cell surface expression data (Fig. 1) along with the qRT-PCR data shown in Fig. 2A demonstrate that Downloaded from microglia express this less investigated receptor. We have previ- ously shown that T cell–derived cytokines such as IFN-g and IL-4 can differentially regulate Fcg receptor subtype expression in microglia (26). These same cytokines regulated IgG-mediated phagocytosis in a manner that could be predicted based on both

the direction of regulation (up or down) and the signal transduc- http://www.jimmunol.org/ tion properties of the Fcg receptors that they regulated (26). Un- like the Fcg receptors, Fca/mR has no canonical cytoplasmic activating or inhibitory signaling domain (33). However, the Fca/ 2/2 FIGURE 7. IgM-induced phagocytosis in WT versus CD18 micro- mR is critical for IgM-mediated phagocytosis in selected periph- A glia. ( ) Representative flow cytometry histogram plots showing fluores- eral immune cell populations (8, 34). Therefore, we hypothesized cent intensity signal for populations of WT or CD182/2 pMG that were incubated with FITC-S. aureus opsonized either by anti-S. aureus IgM (black lines) or isotype control IgM (shaded histograms). Marker gates indicate percentage of anti-S. aureus IgM-induced pMG with FITC fluo- by guest on September 25, 2021 rescent intensity signal greater than background (see Materials and Methods for detail). (B) Bar graph quantifying anti-S. aureus IgM-induced fold change in S. aureus phagocytosis (relative to isotype control IgM) in WT and CD182/2 pMG (see Materials and Methods for details). Total n = 6 (two wells per experimental group from three separate experiments). **p , 0.01 in WT versus CD182/2.

Cytokine-induced regulation of CD11b, CD18, and C3 mRNAs in microglia Given that the rate of IgM-mediated phagocytosis of S. aureus in microglia could be altered by T cell–derived cytokines such as IL-4 (Fig. 2B), as well as the fact that optimal IgM-mediated phagocytosis of S. aureus was dependent on both CR3 (Figs. 6, 7) and C3 (Fig. 8), we sought to determine whether T cell–derived cytokines could alter expression of CR3 components CD11b and CD18 as well as C3. To determine this, we carried out qRT-PCR on RNA extracted from WT pMG that had been treated for 24 h with either IL-4 or IFN-g and quantified expression of steady-state levels of mRNA for CD11b, CD18, or C3. IL-4 significantly reduced expression of CD11b by 62% (Fig. 9A), but had no effect on CD18 or C3 mRNA species (Fig. 9B, 9C). It is possible that this IL-4– FIGURE 8. Quantification of IgM-dependent phagocytosis of S. aureus induced reduction in CD11b expression contributed to the IL-4– 2/2 induced functional downregulation of IgM-mediated phagocytosis in WT versus C3 microglia. Bar graph quantifying anti-S. aureus IgM (A)- or IgG (B)-dependent fold increase in S. aureus phagocytosis (relative of S. aureus (Fig. 2B). In contrast, IFN-g markedly increased the . to isotype control IgM) (see Materials and Methods for detail) in mouse mRNA levels of the C3 ligand by 20-fold (Fig. 9C), but simul- pMG derived from either WT or C32/2 mice. For clarity of comparison, taneously decreased the mRNA levels for CR3 receptor components data are presented as percentage of control of corresponding anti-S. aureus CD11b and CD18 by 63 and 92%, respectively (Fig. 9A, 9B). It IgM or IgG Ab in WT cells. n.s. indicates no significant difference between is possible that these striking, but directionally divergent, IFN-g– WT and C32/2 in (B). Total n = 6–15 samples per group from at least three induced effects on ligand and receptor expression essentially canceled separate experiments. *p , 0.05 versus WT group in (A) (unpaired t test). The Journal of Immunology 5315

FITC-S. aureus in B220+ splenic lymphocytes (8). This functional discrepancy could be due to cell type–specific variation between microglia and splenocytes (35). One key difference may be the level of expression of Fca/mR, as it is much lower in brain than in (8), and also is lower in our cultured microglia than in acutely isolated splenocytes (data not shown). Microglia express and secrete C3 (18), and the latter has been implicated in microglial phagocytosis (18, 36). Microglia also express CR3, the cell surface receptor for C3 (36). Thus, it seemed plausible that complement pathway activation and complement receptors could play a role in the IgM-mediated phagocytosis of S. aureus that we described in Figs. 3 and 4. Our finding that CR3 mediated a significant portion of the IgM-induced response (Figs. 6, 7) is consistent with previous reports implicating CR3 in IgM-mediated phagocytosis of the yeast Cryptococcus neofor- mans (37) and the parasitic protozoa Trypanosoma congolense (38) in macrophages as well as our own data on bone marrow– derived macrophages (Supplemental Fig. 1). In Fig. 6B, we show

that preincubation with anti-CD11b blocking Ab inhibits IgM-, Downloaded from but not IgG-mediated phagocytosis of S. aureus in microglia. This Ig class-specific differential effect of the anti-CD11b blocking Ab points to CR3-dependent elements in the IgM-mediated mecha- nism that may not be in effect for IgG-mediated phagocytosis (or at least not IgG3-mediated phagocytosis). The latter may be re-

lated to class/subclass differences in the efficiency of complement http://www.jimmunol.org/ fixation (29, 39). Another caveat to consider is that complement receptor and Fc receptor coligation can produce cooperative ef- fects (2, 40, 41), so the concentration of target-specific IgG does have an impact on /iC3b-mediated phagocytosis (2, 40, 41). Thus, it is possible that at lower concentrations of anti-S. aureus IgG we might have seen a phagocytosis-inhibiting effect of the anti-CD11b blocking Ab. Nevertheless, the inhibitory effect of the anti-CD11b blocking Ab on IgM-mediated phagocytosis is clear (Fig. 6) and is consistent with our findings in the CD182/2 by guest on September 25, 2021 microglia (Fig. 7). We found that microglial production of C3 is required for op- FIGURE 9. Expression and regulation of CD11b, CD18, and C3 mRNAs in microglia. qRT-PCR on RNA extracted from mouse pMG that timal IgM-, but not IgG-mediated phagocytosis of S. aureus in were either unstimulated (control) or stimulated with 10 ng/ml IL-4 or 10 microglia (Fig. 8). These findings are consistent with our hy- U/ml IFN-g for 24 h. Findings are presented as fold increase in steady- pothesis that complement (and in particular C3) is a key link state levels of CD11b (A), CD18 (B), and C3 (C) mRNA species in the between IgM-opsonized S. aureus and CR3-mediated phagocy- stimulated groups relative to unstimulated controls. Total n = 3–5 samples tosis in microglia. As noted above, microglia can produce and per stimuli from three separate experiments. *p , 0.05 versus corre- release C3. We confirmed robust baseline C3 mRNA expression in sponding unstimulated controls. mouse pMG by qRT-PCR (Fig. 9). C3 release is upregulated by cytokines such as IL-1b, IL-6, and TNF-a (42), and S. aureus that the rate of IgM-mediated phagocytosis would vary in a induces these cytokines in microglia through activation of TLR2 manner directly proportional to Fca/mR expression. However, we (43). Consistent with this, we found in this work that another found that IFN-g markedly reduced Fca/mR expression (Fig. 2A), proinflammatory cytokine, IFN-g, can markedly increase C3 ex- but had no effect on IgM-induced phagocytosis of S. aureus pression (Fig. 9). We considered an alternative explanation that (Fig. 2B). Furthermore, we found that IL-4 induced a significant serum-derived residual active complement factors (including C3), reduction in IgM-mediated phagocytosis of S. aureus (Fig. 2B), possibly cell or extracellular matrix bound (44), might represent but had no effect on Fca/mR mRNA expression (Fig. 2A). Al- an artifactual source of C3 for our pMG in tissue culture. How- though we could not exclude the possibility that these cytokines ever, we purposefully used only heat-inactivated serum to grow might have effects on other components of the cellular phagocytic the microglia and then thoroughly washed and serum starved the machinery, these findings made us question the role of Fca/mRin microglia prior to phagocytosis assay, making this possibility less IgM-induced phagocytosis in microglia. likely. Thus, our data point to microglia themselves as the primary We went on to demonstrate in this study that IgM-induced source of C3 in these studies. phagocytosis was intact in microglia derived from mice with ge- Our data indicate that the effect of genetic deletion of C3 on IgM- netic deletion of Fca/mR (Fig. 5). This finding confirmed that Fca/ mediated phagocytosis of S. aureus was only partial (Fig. 8) and mR did not play a major role in IgM-induced phagocytosis of considerably less robust than the effects induced by inhibiting S. aureus in microglia. Our findings contrast with those of a previ- CR3 (Figs. 6, 7). Approximately 70% of the phagocytic activity ous study demonstrating that 1) transfection of Fca/mR expression persists in microglia from C32/2 mice. One possible explanation vector into pro– line BaF3 induced endocytosis of IgM- is that CR3 is a multifunctional and promiscuous receptor that coated fluorescent beads, and 2) incubation with anti-S. aureus binds to many different ligands in addition to the C3 fragment IgM, but not anti-S. aureus IgG, resulted in phagocytosis of iC3b, including fibrinogen, ICAM-1, and heparin (45–48). The 5316 CR3 MEDIATES IgM-DEPENDENT PHAGOCYTOSIS IN MICROGLIA latter has been shown to facilitate the ligation of IgM in immune 11. Hulett, M. 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