Ca2+ Signaling but Not Store-Operated Ca2+ Entry Is Required for the Function of Macrophages and Dendritic Cells

This information is current as Martin Vaeth, Isabelle Zee, Axel R. Concepcion, Mate of September 23, 2021. Maus, Patrick Shaw, Cynthia Portal-Celhay, Aleena Zahra, Lina Kozhaya, Carl Weidinger, Jennifer Philips, Derya Unutmaz and Stefan Feske J Immunol 2015; 195:1202-1217; Prepublished online 24

June 2015; Downloaded from doi: 10.4049/jimmunol.1403013 http://www.jimmunol.org/content/195/3/1202

Supplementary http://www.jimmunol.org/content/suppl/2015/06/23/jimmunol.140301 http://www.jimmunol.org/ Material 3.DCSupplemental References This article cites 87 articles, 41 of which you can access for free at: http://www.jimmunol.org/content/195/3/1202.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

Ca2+ Signaling but Not Store-Operated Ca2+ Entry Is Required for the Function of Macrophages and Dendritic Cells

Martin Vaeth,*,1 Isabelle Zee,*,1 Axel R. Concepcion,* Mate Maus,* Patrick Shaw,* Cynthia Portal-Celhay,† Aleena Zahra,† Lina Kozhaya,*,† Carl Weidinger,* Jennifer Philips,* Derya Unutmaz,*,† and Stefan Feske*

Store-operated Ca2+ entry (SOCE) through Ca2+ release–activated Ca2+ (CRAC) channels is essential for immunity to infection. CRAC channels are formed by ORAI1 proteins in the plasma membrane and activated by stromal interaction molecule (STIM)1 and STIM2 in the endoplasmic reticulum. Mutations in ORAI1 and STIM1 genes that abolish SOCE cause severe immunodefi- ciency with recurrent infections due to impaired T cell function. SOCE has also been observed in cells of the innate immune Downloaded from system such as macrophages and dendritic cells (DCs) and may provide Ca2+ signals required for their function. The specific role of SOCE in macrophage and DC function, as well as its contribution to innate immunity, however, is not well defined. We found that nonselective inhibition of Ca2+ signaling strongly impairs many effector functions of bone marrow–derived macrophages and bone marrow–derived DCs, including phagocytosis, inflammasome activation, and priming of T cells. Surprisingly, however, macrophages and DCs from mice with conditional deletion of Stim1 and Stim2 genes, and therefore complete inhibition of SOCE, showed no major functional defects. Their differentiation, FcR-dependent and -independent phagocytosis, phagolysosome fusion, http://www.jimmunol.org/ cytokine production, NLRP3 inflammasome activation, and their ability to present Ags to activate T cells were preserved. Our findings demonstrate that STIM1, STIM2, and SOCE are dispensable for many critical effector functions of macrophages and DCs, which has important implications for CRAC channel inhibition as a therapeutic strategy to suppress pathogenic T cells while not interfering with myeloid cell functions required for innate immunity. The Journal of Immunology, 2015, 195: 1202–1217.

2+ 2+ a influx across the plasma membrane through Ca 1,4,5-triphosphate (IP3), opening of IP3 receptor channels in the release–activated Ca2+ (CRAC) channels is an important membrane of the endoplasmic reticulum (ER), and release of Ca2+ C signaling pathway for the activation of immune cells (1). from ER stores. Engagement of other ITAM motif–containing by guest on September 23, 2021 In T lymphocytes, where this pathway is best characterized, immunoreceptors and G protein–coupled receptors on immune CRAC channels are activated after TCR stimulation that results in cells also activates PLCg and PLCb, respectively, and leads to IP3 the activation of phospholipase C (PLC)g, production of inositol production (1). Ca2+ release from the ER results in a transient 2+ 2+ increase in intracellular Ca concentrations ([Ca ]i) and a de- crease in ER Ca2+ concentrations. The latter causes the opening of *Department of Pathology, New York University School of Medicine, New York, NY 10016; and †Department of Medicine, New York University School of Medicine, store-operated CRAC channels in the plasma membrane via the New York, NY 10016 activation of stromal interaction molecule (STIM)1 and STIM2, 1M.V. and I.Z. contributed equally to this work. which are single-pass transmembrane proteins located in the ER 2+ Received for publication December 3, 2014. Accepted for publication May 26, 2015. membrane. Dissociation of Ca from the ER luminal part of This work was supported by National Institutes of Health Grants AI097302 (to S.F.), STIM proteins results in their translocation to ER–plasma mem- AI065303 (to D.U.), and AI087682 (to J.P.) and by postdoctoral fellowships from the brane junctions and binding to ORAI1, the pore-forming subunit National Multiple Sclerosis Society (to P.S.), the Alfonso Martin Escudero Founda- tion (to A.R.C.), and the Deutsche Forschungsgemeinschaft (Grant We 5303/1-1 to of the CRAC channel (2–4). Opening of ORAI1 results in store- 2+ 2+ C.W. and Grant VA 882/1-1 to M.V.). operated Ca entry (SOCE), thus called because this form of Ca 2+ Address correspondence and reprint requests to Prof. Stefan Feske, Department of influx is regulated by the ER Ca concentrations (5). SOCE has Pathology, Experimental Pathology Program, New York University School of Med- been demonstrated not only in T cells but also in many other types icine, 550 First Avenue, Smilow 316, New York, NY 10016. E-mail address: [email protected] of immune cells, including B cells, mast cells, macrophages, dendritic cells (DCs), and neutrophils (1). The online version of this article contains supplemental material. CRAC channels play an important role for immunity to infection, Abbreviations used in this article: AIHA, autoimmune hemolytic anemia; 2-APB, 2-aminoethoxydiphenylborate; AUC, area under the curve; BCG, bacillus Calmette– as patients with inherited mutations in ORAI1 and STIM1 genes Gue´rin; BMDC, bone marrow–derived dendritic cell; BMDM, bone marrow–derived that abolish SOCE suffer from SCID-like disease (6–8), which 2+ 2+ macrophage; [Ca ]i, intracellular Ca concentration; CASR, calcium-sensing re- necessitates hematopoietic stem cell transplantation. These ceptor; CRAC, Ca2+ release–activated Ca2+; DC, dendritic cell; ER, endoplasmic reticulum; FlaA, Legionella pneumophila flagellin; IP3, inositol 1,4,5-triphosphate; patients have recurrent and chronic infections with viruses, bac- LAMP, lysosomal-associated membrane protein; LFn, N-terminal domain of Bacillus teria, and fungal pathogens that have been attributed to impaired anthracis lethal factor; MFI, mean fluorescence intensity; MOI, multiplicity of in- fection; mRBC, mouse RBC; MSU, monosodium urate; NLRP, NOD-like receptor T cell function because of severely impaired proliferation and family, pyrin domain–containing; PA, recombinant anthrax protective Ag; PLC, cytokine production of patient T cells in vitro. T cell–specific 2+ phospholipase C; PRR, pattern recognition receptor; SOCE, store-operated Ca deletion of Stim1 gene expression in mice impairs immunity to entry; STIM, stromal interaction molecule; TG, thapsigargin; WT, wild-type. Mycobacterium tuberculosis (9), and deletion of both Stim1 and + Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 Stim2 compromises antiviral immunity due to impaired CD4 and www.jimmunol.org/cgi/doi/10.4049/jimmunol.1403013 The Journal of Immunology 1203

CD8+ T cell responses (10). In contrast to the well-documented abnormalities in the development of macrophages and DCs in vivo function of CRAC channels in T cells, their role in innate immune or the differentiation of bone marrow–derived macrophages responses is not well defined, and it is unclear whether defects in (BMDMs) and bone marrow–derived DCs (BMDCs) in vitro. myeloid cells contribute to the immunodeficiency of ORAI1- and Despite abolished SOCE, murine macrophages showed normal STIM1-deficient patients. FcR-dependent and independent phagocytosis and maturation of In macrophages, intracellular Ca2+ was shown to regulate sev- phagolysosomes. Furthermore, the production of cytokines in re- eral cell functions such as the production of TNF-a and NO (11, sponse to various pattern recognition receptor (PRR) ligands and 12). FcR-dependent and -independent phagocytosis by macro- the activation of the NLRP3 and NLRC4 inflammasomes were phages is associated with intracellular Ca2+ transients (13–16). normal in STIM1/STIM2-deficient DCs and macrophages. Lastly, Whether phagocytosis requires cytosolic Ca2+ signals, however, is the Ag-presenting function and the subsequent activation of cog- controversial and various studies buffering extra- and intracellular nate T cells remained intact in SOCE-deficient BMDCs. In con- Ca2+ have come to different conclusions (14–17). These early trast, many effector functions of macrophages and DCs, including studies precede the identification of ORAI1, STIM1, and STIM2 phagocytosis, NLRP3 inflammasome activation, and Ag presen- as components of the CRAC channel, thus precluding direct ge- tation, were impaired when Ca2+ signaling was inhibited with netic analysis of how SOCE controls phagocytosis. More recently, nonselective Ca2+ channel modulators or Ca2+ chelating agents. peritoneal macrophages from Stim12/2 mice were reported to have Collectively, our findings demonstrate that Ca2+ signaling, but not a phagocytosis defect (18). Following phagocytosis, phagosomes SOCE via CRAC channels, is required for key effector functions fuse with lysosomes in a process called phagolysosome fusion or of macrophages and DCs. phagosome maturation, which is required for destruction of Downloaded from phagocytosed pathogens. There is evidence that phagosome matu- Materials and Methods ration is dependent on Ca2+ (19–21), although other studies dem- Mice onstrated that this process is Ca2+-independent or even inhibited by fl/fl fl/fl Ca2+ (22, 23). The role of SOCE in phagosome maturation, similar The generation of Stim1 and Stim2 mice was described previously to that in phagocytosis, remains largely unknown. (41). Mice were further crossed to Mx1-Cre (B6.Cg-Tg(Mx1-cre)1Cgn/J, 2+ The Jackson Laboratory, strain 003556) or Vav1-Cre (B6.Cg-Tg(Vav1-cre) In DCs, Ca was reported to promote activation and maturation A2Kio/J, The Jackson Laboratory, strain 008610) mice (42). OT-II http://www.jimmunol.org/ in vitro (24–26) and to play a role in DC responses to TLR ligands mice (C57BL/6-Tg(TcraTcrb)425Cbn/J, The Jackson Laboratory, strain a b or bacteria (27–34). IP3 or LPS stimulation of mouse bone marrow– 004194) crossed to congenic CD45.1 mice (B6.SJL-Ptprc Pepc /BoyJ, derived CD11c+ DCs were shown to induce SOCE and Ca2+ The Jackson Laboratory, strain 002014) were a gift from Dr. Adrian Erlebacher (New York University School of Medicine). All mice were currents resembling ICRAC in T cells (25, 35). Inhibition of SOCE 2+ maintained on a C57BL/6 genetic background. Animals were used be- and Ca currents by the nonselective inhibitor SKF-96365 de- tween 6 and 16 wk of age. Inducible ablation of Stim1 and Stim2 genes in creased the LPS-induced expression of TNF-a and the CCL21- Stim1fl/flStim2fl/fl Mx1-cre mice was carried out by injecting mice three dependent migration of DCs while simultaneously increasing times with 400 mg poly(I:C) (Sigma-Aldrich) every other day i.p. as de- phagocytosis (35). This is consistent with the recently reported scribed before (43). Efficacy of Stim1 and Stim2 deletion was confirmed by immunoblotting and measurements of SOCE. All experiments were con- by guest on September 23, 2021 role of CRAC channels in the activation of human monocyte- ducted in accordance with protocols approved by the Institutional Animal derived DCs in vitro (36). These largely inhibitor-based studies Care and Use Committee of New York University Langone Medical suggest that in vitro differentiated human and mouse DCs require Center. SOCE, but as for macrophages, the precise role of SOCE in DC Media and cell lines maturation and function remains poorly defined. Ca2+ signals have been implicated in the regulation of NOD-like Complete DMEM (supplemented with 10% heat-inactivated FCS, 1% nonessential amino acids, 1% sodium pyruvate, 1% penicillin-streptomycin, receptor family, pyrin domain–containing (NLRP)3 inflammasome and 1% L-glutamine [all CellGro]) containing 10% CMG14-12 cell su- function in myeloid cells (37). The NLRP3 inflammasome is pernatant was used to differentiate BMDMs as described (44). Complete activated by various stimuli, including viruses, bacterial toxins, RPMI 1640 (RPMI 1640 supplemented with 10% heat-inactivated FCS, cholesterol, and monosodium urate (MSU) crystals, which re- 2mML-glutamine, 1 mM pyruvate, 50 mM 2-ME, 100 U/ml penicillin, sult in caspase 1–dependent cleavage of pro–IL-1b and pro–IL-18 100 mg/ml streptomycin [all CellGro]) was used to differentiate BMDCs and maintain T cell cultures. The CMG14-12 cell line was a gift from and secretion of both proinflammatory cytokines. Activation of Dr. Ken Cadwell (New York University School of Medicine). CMG14-12 the NLRP3 inflammasome by ATP and other stimuli was reported cells were grown in complete a-MEM and cell culture supernatant was to require Ca2+ signaling, as inhibition of Ca2+ release from the harvested, sterile filtered through a 0.45-mm filter unit (Millipore), and ER and blocking extracellular Ca2+ influx inhibited NLRP3 tested as previously described (44). 2+ inflammasome function, presumably by preventing Ca -induced Reagents for cell culture mitochondrial damage (38). Extracellular Ca2+, which is increased BMDMs, BMDCs, and T cells were treated with EGTA (2.2 mM final at sites of infection and inflammation and acts as a danger signal, concentration, Sigma-Aldrich), 2-aminoethoxydiphenylborate (2-APB, can also activate the NLRP3 inflammasome directly by binding to 50 mM, Calbiochem), BAPTA-AM (10 mM, Invitrogen), thapsigargin the calcium-sensing receptor (CASR) in the plasma membrane, (TG, 1 mM, Calbiochem), LPS-EB (1–5 mg/ml; Escherichia coli which is a G protein–coupled receptor that mediates the produc- 0111:B4, InvivoGen), curdlan (20–50 mg/ml, InvivoGen), zymosan A tion of IP and Ca2+ release from ER stores (39, 40). However, (25–100 mg/ml, Sigma-Aldrich), CpG (100 nM, Invivogen), 5 or 10 3 multiplicities of infection (MOI) bacillus Calmette–Gue´rin (BCG) (45), none of these studies directly addressed whether modulation of imiquimod (1 mg/ml, InvivoGen), PMA (10 ng/ml, Calbiochem), ionomycin 2+ [Ca ]i via SOCE is involved in the regulation of NLRP3 (0.2 mM, Invitrogen), ATP (3–5 mM, InvivoGen), MSU crystals (150 mg/ml, inflammasome activation. Invitrogen), FlaTox (a combination of 5 mg/ml N-terminal domain of In this study, we investigated the specific role of SOCE in Bacillus anthracis lethal factor [LFn]–Legionella pneumophila flagellin [FlaA] and 10 mg/ml recombinant anthrax protective Ag [PA], a gift of Dr. macrophage and DC function. By using macrophages and DCs Russell Vance, University of California, Berkeley, Berkeley, CA) as indicated. from mice with conditional deletion of Stim1 and Stim2 genes on an inbred C57BL/6 genetic background, we show that macro- Differentiation of BMDMs and BMDCs phages and DCs lack SOCE completely in the absence of STIM1 Femurs and tibiae of mice were removed and bone marrow was flushed out and STIM2. However, abolished SOCE did not result in gross using a 0.45-mm-diameter needle and washed twice as described previously 1204 STORE-OPERATED Ca2+ ENTRY AND MACROPHAGE AND DC FUNCTION

(46). For BMDMs, 5 3 106 bone marrow cells were cultured in 10 ml Immunoblot analysis DMEM containing 10% CMG14-12 cell supernatant for 5 or 6 d, resulting in .90% CD11b+F4/80+ cells. For BMDCs, 2.5 3 106 bone marrow cells Cells were harvested and resuspended in RIPA lysis buffer (50 mM Tris, were cultured RPMI 1640 medium supplemented with 25 ng/ml rGM-CSF 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM (PeproTech) for 8 d resulting in .90% CD11b+CD11c+ cells. EDTA, 1 mM PMSF, 1 mM sodium orthovanadate, and complete protease inhibitor mixture [Sigma-Aldrich]) and sonicated on ice for 10 min. For Isolation of primary peritoneal macrophages Western blot analysis, 50–100 mg total protein was fractionated on a 12% SDS-PAGE and transferred onto nitrocellulose membrane. Membranes Mice were injected i.p. with 1 ml 1% thioglycollate (Difco) in PBS and were incubated with monoclonal goat anti-mouse actin (C4, Santa Cruz sacrificed 3 d later. Abdominal skin was removed (leaving the peritoneum Biotechnology), rabbit anti-mouse caspase-1 p10 (C-20, Santa Cruz Bio- intact), animals were injected with 10 ml sterile PBS, and the peritoneum technology), rabbit anti-mouse anti–IL-1b (D4T2D, Cell Signaling Tech- was massaged to dislodge any attached cells. Lavage fluid was extracted nology), and a custom-made polyclonal rabbit Ab against the C-terminal using a 10 ml syringe and the resulting cell suspension was washed once. domain of STIM1 (8). For detection, peroxidase-coupled secondary anti-goat Cells were resuspended in complete DMEM and plated overnight on tissue or anti-rabbit Abs (Sigma-Aldrich) and the ECL system (Thermo Scientific), culture plates. Nonadherent cells were removed and the remaining adherent or anti-goat IRDye 800CW and anti-rabbit IRDye 680LT Abs and the Od- cells were mainly CD11b+ myeloid cells. yssey Fc Western imaging system (LI-COR Biosciences) were used.

Measurements of intracellular Ca2+ levels Phagocytosis assays using beads 2+ For FcR-independent phagocytosis, BMDMs or BMDCs were plated in Analysis of [Ca ]i in BMDMs and BMDCs by fluorescence microscopy was performed as previously described (47). Briefly, cells were loaded 96-well plates and preincubated with or without 2.2 mM EGTA, 50 mM 2+ m with 2 mM Fura-2-AM (Molecular Probes) for 30 min and [Ca ] was 2-APB, or 10 M BAPTA-AM. After 30 min, yellow-green fluorescent (505/ i 515) carboxylate-modified microspheres (Invitrogen) were added in various analyzed using time-lapse imaging on an IX81 epifluorescence microscope bead-to-cell ratios (1, 5, 25). For FcR-dependent phagocytosis, BMDMs and Downloaded from (Olympus). For measurements of SOCE, baseline [Ca2+] was acquired in i BMDCs were prepared as described above and incubated with FITC-labeled nominally Ca2+-free Ringer solution (155 mM NaCl, 4.5 mM KCl, 3 mM IgG-coated latex beads (Cayman Chemical) at different bead-to-cell ratios MgCl ,10mMD-glucose, and 5 mM Na-HEPES) followed at the indicated 2 (1,5,25).After2hofculture,uptake of fluorescent beads by CD11b+F4/80+ times by stimulation with 1 mM TG (EMD Millipore) or 0.2 mMionomycin BMDMs or CD11c+ BMDCs, respectively, was analyzed by flow cytometry. to deplete ER stores. To induce Ca2+ influx, cells were perfused with To compare different phagocytosis experiments, the changes in mean fluo- Ca2+-containing Ringer solution (155 mM NaCl, 4.5 mM KCl, 2 mM rescence intensity (MFI) values (MFIbeads 2 MFIno beads) were calculated. CaCl2, 1 mM MgCl2,10mMD-glucose, and 5 mM Na-HEPES). For For phagocytosis measurements using fluorescence microscopy, 1 3 105

2+ http://www.jimmunol.org/ measurements of [Ca ]i in response to various PRR ligands, BMDCs were BMDMs were grown on eight-well chamber slides (LabTek) overnight. stimulated with 5 mg/ml LPS-EB (tlrl-eblps, InvivoGen), 20 mg/ml curdlan Cells were treated for 30 min with 10 mM BAPTA-AM or left untreated. (InvivoGen), 100 mg/ml zymosan (Sigma-Aldrich), 1 mg/ml imiquimod Subsequently, BMDMs were incubated with either carboxylate-modified (InvivoGen), or 100 nM CpG (InvivoGen) in 2 mM Ca2+ Ringer solution. 2+ 1-mm yellow-green latex beads (2% solid, Invitrogen) at a 10:1 bead-to- After 420 s, SOCE was induced with 0.2 mM ionomycin. [Ca ]i was cell ratio or FITC-labeled IgG beads (Cayman Chemical) according to the measured as the ratio of fluorescence emission at 510 nm following ex- manufacturers’ protocols. Cells and beads were centrifuged at 300 3 g for citation at 340 and 380 nm (F340/F380) and analyzed using SlideBook 1 min to synchronize the initiation of phagocytosis and after 30 min imaging software v4.2 (Olympus). Alternatively, F340/F380 ratios were washed three times with PBS. Cells were fixed with 4% paraformalde- measured in Fura-2–loaded cells with a FlexStation 3 multi-mode micro- hyde for 30 min and nuclei were stained with 2.5 mg/ml Hoechst 33342 plate reader (Molecular Devices) (48). BMDMs and BMDCs were plated (Molecular Probes). Images were captured using a Nikon Eclipse TiE/B on poly-L-lysine–coated translucent 96-well plates (BD Falcon) for 15 min, automated fluorescence microscope with Photometrics HQ2 mono- by guest on September 23, 2021 2+ washed, and incubated in 0 mM Ca Ringer solution with or without chrome digital camera. Z-stack images (360) were acquired, and the 2.2 mM EGTA, 50 mM 2-APB, or 10 mM BAPTA-AM for 30 min before number of latex beads or FITC+ vacuoles per cell was quantified using 2+ 2+ measurements of [Ca ]i.Ca influx was induced by stimulating cells with NIS-Elements DUO software. At least 5 fields per sample with an av- 1 mM TG as described above or by FcR cross-linking. For the latter, erage cell number of 15 per field were analyzed. BMDMs were incubated with 10 mg/ml rat anti-mouse FcgIIR/FcgIIIR (CD16/CD32, clone 2.4G2, BD Pharmingen) for 30 min followed by Phagocytosis assay using opsonized mouse RBC and 20 mg/ml goat anti-rat IgG (BioLegend). Following stimulation of cells in Staphylococcus aureus 2+ Ca -free buffer, an equal volume of 2 mM CaCl2 Ringer solution was added to cells (to obtain a final extracellular [Ca2+] of 1 mM). Ca2+ store Fresh mouse RBCs (mRBCs) were extracted from the blood of wild-type depletion was measured as the area under the curve (AUC) after TG or (WT) C57BL/6 mice using Ficoll-Plaque gradient centrifugation and ionomycin stimulation in Ca2+-free buffer, and the peak of Ca2+ influx washed once with PBS. mRBC solution (200 ml) was opsonized with 25 ml (SOCE) was measured as the maximum F340/380 value after re-addition anti-mouse RBC Ab (34-3C, Abcam) for 1 h and labeled with 8 ml anti– of extracellular Ca2+ minus the baseline F340/380 value at the beginning Ter119-PE Ab. After washing twice with PBS, 2, 20, or 200 opsonized mRBCs per cell were incubated with BMDMs or peritoneal macrophages of the experiment. for 3 h. Phagocytosis was analyzed by measuring Ter119+ cells using flow Flow cytometry cytometry. GFP-expressing S. aureus (Newman strain) was a gift of Dr. Victor Torres (New York University School of Medicine) (49). S. aureus Cells were washed once in flow cytometry staining buffer (13 PBS, was harvested during the logarithmic growth phase and incubated at 1, 10, 0.1% BSA) before blocking with anti-FcgRII/FcgRIII (2.4G2, BD or 50 MOI with BMDMs for 2 h. Phagocytosis was analyzed testing the + Pharmingen) for 5 min. Staining of surface molecules was performed uptake of GFP-expressing bacteria in CD11b cells by flow cytometry and in FACS buffer (13 PBS, 0.5% FCS) for 20 min in the dark using the fluorescence microscopy using an automated Nikon Eclipse TiE/B system following anti-mouse Abs: anti–CD11b-PE-Cy7 or anti–CD11b-allophycocyanin with Photometrics HQ2 monochrome digital camera and NIS-Elements (clone M1/70), anti–F4/80-PerCP-Cy5.5 (BM8), anti–CD11c-eFluor 450 DUO software package. (N418), anti–MHC class II-PE or anti–MHC class II-eFluor 450 (I-A/I-E, Lysosomal trafficking assays M5/114.15.2), anti–CD86-allophycocyanin (GL1), and anti–IFN-g- allophycocyanin (XMG1.2, all eBioscience). For human PBMCs, the GFP-expressing S. aureus (Newman strain) were harvested during their following Abs were used: anti–CD3-PE-Cy7 (UCHT1), anti–CD14-Alexa logarithmic-growth phase and incubated with BMDMs at an MOI of 10 for Fluor 700 (HCD14), anti–CD19-PE-Cy7 (HIB19), anti–CD11b-brilliant 60 and 120 min. LysoTracker Red (Invitrogen) was added to BMDMs blue 605 (M1/76), anti–HLA-DR-allophycocyanin-Cy7 (L243), and anti– 30 min before the end of the incubation period and extracellular S. aureus CD303-PerCP-Cy5.5 (201A, all BioLegend). Stained cells were analyzed was lysed with lysostaphin (Sigma-Aldrich). Cells were fixed with 4% using a FACS LSR II flow cytometer (BD Biosciences) and FlowJo soft- paraformaldehyde in PBS, and confocal images were acquired with an ware (Tree Star). For intracellular cytokine staining, OT-II+ CD4+ T cells inverted Zeiss LSM 510 laser scanning confocal microscope and a 603,1.45 wererestimulatedfor6hwith20nMPMA(Calbiochem)and1mM numerical aperture oil-immersion objective (Zeiss). Image analysis was done ionomycin (Invitrogen) in the presence of 3 mg/ml brefeldin A (eBioscience). using ImageJ software (National Institutes of Health). The percentage of After surface staining, cells were fixed with IC fixation buffer and per- phagocytosed S. aureus colocalizing with LysoTracker Red was determined meabilized using 13 Perm buffer (both eBioscience) according to the by blinded counting. To study trafficking of phagocytosed mycobacteria to manufacturer’s instructions. lysosomes, BMDMs were infected with 10 MOI of GFP-expressing BCG (an The Journal of Immunology 1205 attenuated strain of Mycobacterium bovis) (45) for 3 h, then washed three immune responses require SOCE and that abolished SOCE in times with media and incubated another 24 h in RPMI 1640 medium at patients with loss-of-function or null mutations in ORAI1 and 37˚C. Cells were washed once and fixed with 4% paraformaldehyde in PBS STIM1 genes may contribute to their immunodeficiency. We and for 30 min, permeabilized with 0.1% saponin in PBS for 5 min, and incubated with polyclonal rabbit anti–lysosomal-associated membrane protein others had previously described that ORAI1 and STIM1-deficient (LAMP-1) Ab (Abcam). Images were captured using a Nikon Eclipse TiE/B patients have normal numbers of monocytes, neutrophils, baso- automated fluorescent microscope with Photometrics HQ2 monochrome phils, and eosinophils in their peripheral blood, suggesting that 3 digital camera. Z-stack ( 60) images were acquired and analyzed using SOCE mediated by CRAC channels is not required for the de- NIS-Elements DUO software as previously described (45). velopment of myeloid cells (7, 8, 51). Accordingly, we found that Cytokine ELISA the frequencies of CD14+CD303– monocytes, CD14–CD303– – + BMDMs or BMDCs (1 3 106/ml) were left untreated or stimulated with conventional DCs, and CD14 CD303 plasmacytoid DCs were 1 mg/ml LPS-EB (tlrl-eblps, InvivoGen), 50 mg/ml curdlan (InvivoGen), comparable in the peripheral blood of a 5-mo-old patient with 25 mg/ml zymosan A (Sigma-Aldrich), 100 nM CpG (InvivoGen), 5 MOI a homozygous R91W loss-of-function mutation in ORAI1 (re- BCG (M. bovis-BCG) (45), or 1 mg/ml imiquimod (InvivoGen) for 16 h. ferred to as ORAI1R91W) (2) and a healthy donor control Cytokine concentrations in supernatants were analyzed with the Ready- (Supplemental Fig. 1). SET-Go! ELISA kits detecting mouse IL-1b, IL-2, IL-6, IL-10, IL-12p40, IL-12p70, IL-23p19, and TNF-a (all eBioscience) according to the Because patients with genetic defects in ORAI1 and STIM1 manufacturer’s instructions and measured at 450 nm with a SpectraMax genes are very rare and surviving patients have leukocyte chi- M5 ELISA reader (Molecular Devices). merism after hematopoietic stem cell transplantation, it is difficult NLRP3 and NLRC4 inflammasome activation to study the role of SOCE in innate immunity in human patients. Various studies have used human or mouse myeloid cell lines or Downloaded from For NLRP3 inflammasome activation, BMDMs and BMDCs were plated at 2+ 3 6 primary cells and investigated Ca signaling either by chelating a density of 1 10 cells per ml, primed with 10 mg/ml LPS for 4 h (with 2+ or without 2.2 mM EGTA, 50 mM 2-APB, or 10 mM BAPTA-AM), and extra- or intracellular Ca (with EGTA or BAPTA) or by using 2+ stimulated for 30 min with 3 mM ATP (InvivoGen). Alternatively, the nonselective Ca channel inhibitors (36, 52–54). Because these NLRP3 inflammasome was activated by stimulation with 150 mg/ml MSU approaches do not specifically inhibit CRAC channels and SOCE, crystals (Invitrogen) for 4 h. For NLRC4 inflammasome activation, cells we decided to use mice with conditional deletion of Stim1 and were incubated for 15 or 30 min with a combination of 5 mg/ml FlaA fused Stim2 genes in hematopoietic cells (41). These mice were gener- http://www.jimmunol.org/ LFn and 10 mg/ml PA, referred to as FlaTox (gift of Dr. Russell Vance, University of California, Berkeley). Following stimulation, cell culture ated on a defined inbred C57BL/6 genetic background to avoid supernatants were harvested and analyzed for IL-1b secretion by ELISA and confounding effects by polymorphisms in genes not related to cells were lysed and analyzed for procaspase-1 cleavage by Western blot. CRAC channel function. First, we ablated STIM protein expres- fl/fl fl/fl fl/fl Ag presentation and T cell activation assay sion in Stim1 Mx1-Cre or Stim1 Stim2 Mx1-Cre mice by poly(I:C) injection (41, 43). To rule out any potential immuno- BMDCs were maturated into APCs with 0.1 mg/ml LPS with or without 10 logical artifacts arising from the transient type I IFN response mM BAPTA-AM for 30 min. After washing, BMDMs or BMDCs were induced by poly(I:C), we also generated Stim1fl/flStim2fl/fl Vav-Cre incubated overnight with 0.5 mg/ml OVA protein (Sigma-Aldrich). OVA- specific CD4+ T cells were isolated from the spleen and lymph nodes of mice, in which Stim1 and Stim2 are conditionally deleted in all OT-II mice using the EasyStep mouse CD4+ T cell enrichment kit hematopoietic cells without any further treatment (55). Hematopoietic by guest on September 23, 2021 (StemCell Technologies). CD4+ T cells were labeled with 2.5 mM Cell- precursor cells from both strains of mice were then differentiated Trace CFSE (Invitrogen) for 5 min and washed twice. Unbound OVA was into BMDMs and BMDCs in vitro. removed from APC cultures before coculturing with CFSE-labeled OT-II CD4+ T cells in 5:1 and 1:1 APC/T cell ratios. After 72 h, proliferation The efficiency of STIM1 and STIM2 deletion in BMDMs from fl/fl fl/fl fl/fl (i.e., CFSE dilution) was analyzed by flow cytometry. As a negative WT, Stim1 Mx1-Cre,andStim1 Stim2 Mx1-Cre mice was control, OT-II T cells were incubated with BMDCs without OVA protein analyzed at the functional level by Ca2+ imaging (Fig. 1A). and, as a positive control, OT-II T cells were stimulated polyclonally using Deletion of STIM1 alone or both STIM1 and STIM2 strongly anti-CD3 and anti-CD28 mAbs (145-2C11 and 37.51, both eBioscience). fl/fl + impaired SOCE in BMDMs from poly(I:C)-injected Stim1 Mx1-Cre For the detection of intracellular IFN-g, OT-II T CD4 cells were restimulated with 20 nM PMA (Calbiochem) and 1 mM ionomycin mice and abolished SOCE in BMDMs from poly(I:C)-injected (Invitrogen) in presence of 3 mg/ml brefeldin A (eBioscience) for 6 h and Stim1fl/flStim2fl/fl Mx1-Cre mice (Fig. 1A and data not shown) analyzed by flow cytometry. when SOCE was induced with TG, an inhibitor of the sarco/ endoplasmic reticulum ATPase (SERCA) pump, or ionomycin, Patient samples which leaks Ca2+ from ER stores. In contrast, deletion of STIM1 Frozen PBMCs from a healthy donor and a SOCE-deficient patient ho- alone or both STIM1 and STIM2 had no significant effect on Ca2+ R91W 3 5 mozygous for the ORAI1 mutation (2) were thawed and 5 10 cells store depletion. The absence of STIM1 protein in BMDMs from were stained for cell surface markers to determine monocyte and DC pop- fl/fl fl/fl fl/fl ulations and analyzed by flow cytometry. Informed consent for the studies Stim1 Mx1-Cre and Stim1 Stim2 Mx1-Cre mice was con- was obtained from the patient’s family in accordance with the Declaration of firmed by Western blotting (Fig. 1B). Despite the severe defect in Helsinki and Institutional Review Board approval of the New York Uni- SOCE, the numbers and immunological phenotype of BMDMs versity School of Medicine. from Stim1fl/fl Mx1-Cre and Stim1fl/flStim2fl/fl Mx1-Cre mice were Statistical analysis comparable to WT controls (Fig. 1C), suggesting that SOCE is not required for the differentiation of BMDMs in vitro. Similar results Experimental data were analyzed with Prism software (GraphPad Soft- were obtained using BMDMs from Stim1fl/flStim2fl/fl Vav-Cre mice, ware), and statistical significance was calculated by a two-tailed paired or which lacked SOCE (Fig. 1D) but similar to WT BMDMs were unpaired Student t test. Differences with p values ,0.05 were considered . + + to be statistically significant. 99% F4/80 Cd11b (Fig.1E).WenexttestedtheeffectsofSTIM1 andSTIM2deletioninBMDCsgeneratedfromStim1fl/flStim2fl/fl Vav-Cre BM cells (Fig. 1F–H). STIM1/STIM2-deficient BMDCs Results showed unaltered Ca2+ store release compared with WT control cells, STIM1 and STIM2 are not required for the differentiation of whereas SOCE was completely absent in response to TG (Fig. 1F) or macrophages and DCs ionomycin (data not shown) stimulation. CRAC channels and STIM1 was shown to mediate SOCE in macrophages (18), DCs STIM1 or STIM2 were suggested to play a role in the differ- (36), and neutrophils (50). It therefore seemed likely that innate entiation and maturation of DCs (25, 36, 56). However, 1206 STORE-OPERATED Ca2+ ENTRY AND MACROPHAGE AND DC FUNCTION Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 1. STIM1 and STIM2 are not required for the differentiation of macrophages and DCs. (A–C) Analysis of BMDMs from poly(I:C)-treated WT, Stim1fl/fl Mx1-Cre, and Stim1fl/flStim2fl/fl Mx1-Cre mice. (A) SOCE in BMDMs was analyzed by time-lapse microscopy. Ca2+ store depletion was induced by 2+ 2+ 2+ TG stimulation in Ca -free buffer and analyzed as AUC220 s–410 s; SOCE was induced by re-addition of 2 mM extracellular Ca and analyzed as peak Ca influx. Data represent three independent experiments. (B) Deletion of STIM1 protein expression. Lysates of BMDMs were separated by SDS-PAGE and incubated with Abs to STIM1 and actin. Shown is one representative experiment of three. n.s., nonspecific. (C) Phenotypic characterization of BMDMs by flow cytometry. (D and E) Analysis of BMDMs differentiated from BM of WT and Stim1fl/flStim2fl/fl Vav-Cre mice. (D) SOCE was measured using a FlexStation 3 plate reader and analyzed as described in (A). (E) Phenotypic characterization of BMDMs was done as described in (C). Data represent three independent experiments. (F–H) Analysis of BMDCs from WT and Stim1fl/flStim2fl/fl Vav-Cre mice. (F)Ca2+ store depletion and SOCE in BMDCs were induced as described in (A) and measured using a FlexStation 3 plate reader. Graphs shown are representative of four independent experiments. (G) Phenotypic characterization of immature BMDCs stained with Abs to CD11c, CD86, and MHC class II and analyzed by flow cytometry. (H) Numbers of immature BMDCs after 8 d in culture starting with 2 3 106 bone marrow cells. Bar graphs represent the mean 6 SEM of nine mice per group. Statistical significance was calculated with an unpaired Student t test. *p , 0.05, **p , 0.005.

we observed normal expression of CD11c, CD86, and MHC class channels mediating these Ca2+ signals remained unclear. Earlier II on immature BMDCs generated from Stim1fl/flStim2fl/fl Vav-Cre studies used mostly Ca2+ chelators to buffer extracellular and mice compared with WT controls (Fig. 1G, 1H). Taken together, cytosolic Ca2+ or nonselective ion channel inhibitors (15, 16). To STIM1 and STIM2 are essential for SOCE but not the differen- investigate the specific contribution of SOCE to FcR-dependent tiation and maturation of BMDMs and BMDCs in vitro. We were and -independent phagocytosis, we analyzed BMDMs (Fig. 2, therefore able to use BMDMs and BMDCs derived from Stim1fl/fl Supplemental Fig. 2) and BMDCs (Supplemental Fig. 3) from Stim2fl/fl Vav-Cre and Stim1fl/flStim2fl/fl Mx1-Cre mice to investigate the Stim1fl/flStim2fl/fl Mx1-Cre and Stim1fl/flStim2fl/fl Vav-Cre mice. For role of SOCE and CRAC channels in DC and macrophage function. comparison, we investigated the effects of extracellular and cy- 2+ 2+ tosolic Ca chelation with EGTA and BAPTA-AM, respectively, Ca signaling but not SOCE is required for phagocytosis in and those of inhibiting Ca2+ influx and ER store release with the macrophages and DCs widely used but nonselective ion channel modulator 2-APB, Ca2+ signals were shown in several studies to be required for which has complex effects on CRAC channels, TRP channels, and 2+ macrophage phagocytosis (13–16), but the source of Ca and the IP3Rs (57–60). The Journal of Immunology 1207 Downloaded from http://www.jimmunol.org/

FIGURE 2. Phagocytosis by BMDMs requires intracellular Ca2+ signaling but not SOCE. (A–C) WT and Stim1fl/flStim2fl/fl Vav-Cre BMDMs were left

2+ by guest on September 23, 2021 untreated (medium) or incubated with 2.2 mM EGTA (A), 10 mM BAPTA-AM (B), or 50 mM 2-APB (C) for 30 min and [Ca ]i was analyzed using 2+ a FlexStation 3 (A–C, left panels). Store depletion (AUC60 s–380 s) and peak Ca influx (peak F340/380) were quantified from three individual mice per group (A–C, right panels). (D and E) WT and Stim1fl/flStim2fl/fl Vav-Cre BMDMs were incubated with 1, 5, and 25 bead-to-cell ratios of carboxylate- modified yellow-green microspheres (D) or FITC-labeled IgG-coated fluorescent beads (E) for 2 h with medium alone or with 2.2 mM EGTA, 10 mM BAPTA-AM, or 50 mM 2-APB as described in (A)–(C). Phagocytosis was quantified by flow cytometry (left panels) and data from three mice per group were compiled as ΔMFI (right panel), where ΔMFI = MFIbeads 2 MFIno beads. Bar graphs represent the mean 6 SEM. Statistical significance was calculated with an unpaired Student t test. *p , 0.05, **p , 0.005, ***p , 0.001.

Deletion of STIM1 and STIM2 in BMDMs and BMDCs com- indicating that these agents perturb the filling state of ER Ca2+ pletely inhibited SOCE after TG stimulation compared with WT stores by interfering with other Ca2+ signaling pathways besides control cells (Fig. 2A–C, Supplemental Fig. 3A–C). Chelation of SOCE. STIM1/STIM2-deficient mice and the nonselective inhib- extracellular Ca2+ with EGTA to prevent Ca2+ influx (Fig. 2A, itors of Ca2+ signaling allowed us to distinguish between the Supplemental Fig. 3A), chelation of cytosolic Ca2+ with BAPTA- specific contributions of SOCE (absent in STIM1/STIM2-deficient AM (Fig. 2B, Supplemental Fig. 3B), and treatment with 2-APB cells), Ca2+ influx via SOCE plus other plasma membrane Ca2+ (Fig. 2C, Supplemental Fig. 3C) reduced the TG-induced Ca2+ channels (inhibited by 2-APB and EGTA), and Ca2+ release from influx in WT cells to a similar degree as did deletion of Stim1 and intracellular stores plus Ca2+ influx (neutralized by BAPTA) to Stim2 genes. No additional reduction of Ca2+ influx was observed BMDM and BMDC function. in STIM1/STIM2-deficient cells after treatment with EGTA, To evaluate the role of SOCE and Ca2+ signals in phagocytosis, BAPTA-AM, or 2-APB compared with control cells. These find- we first analyzed the FcR-independent uptake of fluorescent latex ings indicate that TG-induced SOCE in BMDMs and BMDCs is beads by BMDMs (Fig. 2D, Supplemental Fig. 2A) and BMDCs completely dependent on STIM1 and STIM2. (Supplemental Fig. 3D). Stim1fl/flStim2fl/fl Vav-Cre and WT control In contrast, Stim1 and Stim2 gene deletion had no effect on the BMDMs were left untreated or pretreated with EGTA, BAPTA-AM, release of Ca2+ from ER stores. The integrated Ca2+ signal fol- or 2-APB and incubated with latex beads at different bead-to-cell ratios lowing TG or ionomycin stimulation in Ca2+-free extracellular (1:1, 5:1, and 25:1). Flow cytometric analysis did not show a differ- buffer was comparable in STIM1/STIM2-deficient and WT con- ence in phagocytosis by BMDMs from Stim1fl/fl Mx1-Cre, Stim1fl/fl trol BMDMs (Fig. 2A–C and data not shown) and BMDCs Stim2fl/fl Mx1-Cre (Supplemental Fig. 2A), or Stim1fl/flStim2fl/fl (Supplemental Fig. 3A–C and data not shown), indicating that the Vav-Cre mice (Fig. 2D) compared with WT controls. Likewise, filling of ER Ca2+ stores is intact in the absence of STIM1 and inhibition of Ca2+ influx with 2-APB or EGTA did not substan- 2+ STIM2. In contrast, the [Ca ]i rise following TG-induced ER tially impair phagocytosis of STIM1/STIM2-deficient or WT store depletion was attenuated in cells treated with 2-APB, EGTA, macrophages (Fig. 2D). In contrast, chelation of intracellular and especially BAPTA (Fig. 2A–C, Supplemental Fig. 3A–C), Ca2+ with BAPTA significantly reduced phagocytosis by BMDMs 1208 STORE-OPERATED Ca2+ ENTRY AND MACROPHAGE AND DC FUNCTION

(Fig. 2D). Similar results were obtained using fluorescence mi- Taken together, these findings demonstrate that STIM1 and STIM2 are croscopy to analyze phagocytosis (Fig. 3A, 3B). Furthermore, we not required for FcR-dependent phagocytosis by in vitro–differentiated found that phagocytosis of latex beads in BMDCs is independent and peritoneal macrophages. Treatment of BMDMs with 2-APB of STIM1 and STIM2 (Supplemental Fig. 3D). We next tested the had no effect on the phagocytosis of IgG-coated beads, whereas FcR-independent phagocytosis of bacteria by incubating BMDMs chelation of extracellular Ca2+ with EGTA partially inhibited with live GFP-expressing S. aureus. Bacterial phagocytosis was phagocytosis, and chelation of cytosolic Ca2+ with BAPTA unaltered in BMDMs from Stim1fl/fl Mx1-Cre and Stim1fl/flStim2fl/fl completely inhibited phagocytosis (Fig. 2E). Similar results were Mx1-Cre mice (Supplemental Fig. 2B). Taken together, these data obtained in BMDCs (Supplemental Fig. 3E). The effect of BAPTA show that FcR-independent phagocytosis is regulated by cytosolic on the phagocytosis of IgG-coated beads was more pronounced Ca2+ levels but is independent of SOCE mediated by STIM1 and compared with noncoated beads when analyzed either by flow STIM2. cytometry (Fig. 2D, 2E) or fluorescence microscopy (Fig. 3C, 3D), FcR crosslinking results in STIM1-dependent SOCE that was suggesting that cytosolic Ca2+ signaling may be more critical for shown to be required for FcR-dependent phagocytosis (18). We FcR-dependent phagocytosis. Taken together, our results demon- confirmed that crosslinking of FcgRII/III (CD16/32) results in strate that whereas intracellular Ca2+ signals are required for FcR- rapid SOCE in WT control BMDMs, which was almost com- dependent and -independent phagocytosis by macrophages and pletely abolished in BMDMs from Stim1fl/flStim2fl/fl Vav-Cre DCs, SOCE mediated by STIM1 and STIM2 is dispensable for this BMDMs (Supplemental Fig. 2C). To evaluate whether STIM1 process. and STIM2 are required for FcR-mediated phagocytosis, we in- cubated BMDMs with fluorescent IgG-coated beads (Fig. 2E, Lysosomal trafficking is independent of STIM1 and STIM2 Downloaded from Supplemental Fig. 2D) and Ab-opsonized mouse RBCs Following phagocytosis, endosomes fuse with Golgi apparatus– (Supplemental Fig. 2E). Surprisingly, the phagocytosis of IgG- derived lysosomes that contain hydrolytic enzymes. This process coated beads by BMDMs from Stim1fl/fl Mx1-Cre mice, Stim1fl/fl of phagolysosome fusion is required for the degradation of Stim2fl/fl Mx1-Cre mice (Supplemental Fig. 2D), and Stim1fl/flStim2fl/fl phagocytosed pathogens and the production of pathogen-derived Vav-Cre mice (Fig. 2E) was comparable to WT controls. Normal peptides presented on MHC class II molecules. The involvement 2+ phagocytosis of Ab-opsonized mRBCs was also observed in BMDMs of Ca signals in lysosomal trafficking and phagolysosome fusion http://www.jimmunol.org/ (Supplemental Fig. 2E) and, importantly, in primary peritoneal mac- is debated (19–23), and the specific role of SOCE in this process rophages from poly(I:C)-treated Stim1fl/fl Mx1-Cre or Stim1fl/flStim2fl/fl has not been investigated. We therefore analyzed intracellular Mx1-Cre mice compared with WT controls (Supplemental Fig. 2F). trafficking and colocalization of live GFP-expressing S. aureus by guest on September 23, 2021

FIGURE 3. Intracellular Ca2+ signaling but not SOCE is required for FcR-dependent and FcR- independent phagocytosis in BMDMs. BMDMs (1 3 105) from WT and Stim1fl/flStim2fl/fl Vav-Cre mice were treated with 10 mM BAPTA-AM for 30 min or left untreated and incubated with either carboxylate- modified yellow-green microspheres at a 10:1 bead- to-cell ratio (A and B) or FITC-labeled IgG-coated beads (C and D). After 30 min, cells were washed three times with PBS, fixed, and nuclei were counterstained with Hoechst 33342 dye. Phagocy- tosis was analyzed by fluorescence microscopy by measuring the number of beads per cell (A and B)or the number of FITC+ vacuoles per cell (C and D) from .50 cells per condition. Original magnifica- tion in (A) and (C) 360. Bar graphs in (C) and (D) represent the mean 6 SEM. Statistical significance was calculated with an unpaired Student t test. **p , 0.01, ***p , 0.001. The Journal of Immunology 1209 with lysosomes in BMDMs from WT and Stim1fl/flStim2fl/fl Mx1-Cre and zymosan stimulation were largely absent in STIM1/STIM2- mice (Fig. 4A, 4B). The phagocytosis of S. aureus was not deficient BMDCs compared with WT cells (although differences impaired in STIM1/STIM2-deficient BMDMs, as the number of were not statistically significant), suggesting that they may be GFP+ bacteria per cell was comparable to WT control cells mediated by SOCE (Supplemental Fig. 4B, 4C). Similar obser- (Fig. 4C), consistent with results obtained by flow cytometry vations were made in BMDMs (data not shown). Taken together, (Supplemental Fig. 2B). We did not detect differences in the PRR ligands such as LPS and zymosan induce weak Ca2+ influx distribution pattern (Fig. 4A) or extent (Fig. 4B) of colocalization that may be partially mediated by SOCE, but these signals are between S. aureus and LysoTracker-positive vesicles in WT and dispensable for PRR-induced cytokine production in BMDMs Stim1fl/flStim2fl/fl Mx1-Cre BMDMs. and BMDCs.

Using a second pathogen, we incubated live GFP-expressing BCG 2+ (BCG-GFP), an attenuated vaccine strain of M. bovis,for3hwith Cytosolic Ca but not SOCE is required for NLRP3 and BMDMs from WT, Stim1fl/fl Mx1-Cre,andStim1fl/flStim2fl/fl Mx1-Cre NLRC4 inflammasome activation mice (Fig. 4D, 4E). After removal of extracellular bacteria, Another mechanism by which innate immune cells produce py- BMDMs were cultivated for another 24 h before colocalization of rogenic cytokines is through activation of inflammasomes. Several GFP and lysosomal-associated membrane protein (LAMP)-1, studies have linked Ca2+ signaling to the activation of NLRP3 and a widely used marker for lysosomes, was evaluated by confocal NLRC4 inflammasomes (38–40, 68, 69). Ca2+ release from the ER microscopy (Fig. 4D, 4E) (45). Quantification of the LAMP-1 (38–40, 53) and binding of extracellular Ca2+ to the CASR (38, signal surrounding the internalized GFP-tagged BCG showed a 40) were described to modulate NLRP3 activation. However, most similar extent of colocalization in BMDMs from Stim1fl/fl Mx1-Cre of these studies relied on the use of Ca2+ chelators or nonselective Downloaded from and Stim1fl/flStim2fl/fl Mx1-Cre mice compared with WT controls ion channel inhibitors (53, 54, 68, 69), and the specific role of (Fig. 4E), indicating that intracellular trafficking of mycobac- SOCE in inflammasome activation has not been investigated. To teria into LAMP-1+ organelles was not altered. Taken together, elucidate whether Ca2+ signals and SOCE are required for NLRP3 our findings show that SOCE mediated by STIM1 and STIM2 is activation, we first stimulated WT BMDMs with LPS for 4 h dispensable for phagolysosome fusion in macrophages. followed by stimulation with ATP for 30 min. WT BMDMs se-

creted readily detectable amounts of IL-1b, which were signifi- http://www.jimmunol.org/ Cytokine production by BMDMs and BMDCs does not require cantly reduced by treatment of cells with 2-APB and abolished by SOCE chelation of intracellular Ca2+ with BAPTA (Fig. 6A), indicating With degradation of internalized pathogens, so-called “danger that Ca2+ signals are indeed required for NLRP3 function. Che- signals” or pathogen-associated molecular patterns are released lating extracellular Ca2+ with EGTA, in contrast, had no effect on and trigger maturation and activation of myeloid cells. Various IL-1b production. PRRs were reported to induce Ca2+ signals (27–29, 31, 33, 61– ATP was shown to activate the NLRP3 inflammasome by 63), and the involvement of SOCE therein has been discussed (29, opening P2X7 receptor channels that, besides causing K+ efflux, 31, 36). Ca2+ signals are well known to mediate the activation of mediate Ca2+ influx into cells (70). In addition to P2X7 receptors, transcription factors such as NFAT and NF-kB and thereby the ATP binds to G protein–coupled P2Y receptors that induce SOCE by guest on September 23, 2021 expression of cytokines (64). To evaluate the role of SOCE in via activation of PLCb and production of IP3. We therefore regulating cytokine production in myeloid cells, we stimulated measured Ca2+ influx in STIM1/STIM2-deficient BMDMs and BMDMs and BMDCs from Stim1fl/flStim2fl/fl Vav-Cre and WT BMDCs following stimulation with 5 mM ATP (Fig. 6B, 6G). We mice with various synthetic or natural PRR ligands that have been observed moderately reduced Ca2+ influx in STIM1/STIM2- shown to induce Ca2+ signals, including LPS (detected by CD14/ deficient cells compared with WT cells, which is likely due to TLR4) (29, 65, 66), CpG (TLR9) (27, 28), imiquimod (TLR7) (62, impaired P2Y receptor–induced SOCE. To analyze whether SOCE 63), curdlan and zymosan (both recognized by Dectin-1 and/or is required for NLRP3 activation, we next tested BMDMs from TLR2) (32–34, 67), and live BCG (presumably triggering several WT and Stim1fl/flStim2fl/fl Vav-Cre mice. STIM1/STIM2-deficient PRRs) (Fig. 5). Zymosan and curdlan are likely to induce SOCE, as BMDMs produced copious amounts of IL-1b comparable to 2+ their binding to Dectin-1 activates Syk and PLCg, resulting in IP3 WT cells (Fig. 6C) despite reduced ATP-induced Ca influx. production and ER store release (34, 67). For other ligands such as Consistent with normal IL-1b secretion, BMDMs from Stim1fl/fl LPS, CpG, and imiquimod, the source of Ca2+ is not known. Sur- Mx1-Cre and Stim1fl/flStim2fl/fl Mx1-Cre mice showed normal prisingly, BMDMs and BMDCs from Stim1fl/flStim2fl/fl Vav-Cre cleavage of procaspase-1 into (active) caspase-1 (p10) after LPS produced similar amounts of TNF-a, IL-6, IL-10, IL-12p70, and ATP stimulation that was comparable to WT BMDMs IL-23p19, IL-2, and IL-12/23p40 as WT cells when stimulated (Fig. 6D). To distinguish between a potential role of SOCE in the with LPS, CpG, zymosan, curdlan, BCG, or imiquimod for 16 h priming and activation of the NLRP3 inflammasome, we analyzed (Fig. 5), indicating that STIM1 and STIM2, and thereby SOCE, the intracellular pro–IL-1b levels during the priming phase with are dispensable for PRR-induced cytokine production in myeloid LPS and the cleavage-induced decrease of intracellular pro–IL-1b cells. To investigate whether PRR ligands induce Ca2+ signals upon ATP stimulation (Fig. 6E). We found comparable expression andwhetherthesedependonSTIM1andSTIM2,wemeasured levels of pro–IL-1b in BMDMs from Stim1fl/flStim2fl/fl Vav-Cre Ca2+ influx in WT and STIM1/STIM2-deficient BMDCs in re- and WT mice during the entire LPS stimulation period of 16 h sponse to LPS, CpG, zymosan, curdlan, or imiquimod stimula- (Fig. 6E, left panel) and a comparable decrease of intracellular tion (Supplemental Fig. 4). In WT BMDCs, we observed small pro–IL-1b in WT and STIM1/STIM2-deficient BMDMs after ATP 2+ transient rises in [Ca ]i in response to LPS and zymosan, treatment (Fig. 6E, right panel). whereas stimulation with curdlan, imiquimod, or CpG failed to IL-1b production was also normal after treatment of STIM1/ result in Ca2+ increases. Even after LPS and zymosan stimula- STIM2-deficient BMDMs with MSU crystals, another danger tion, only few cells within the entire cell population exhibited signal that activates the NLRP3 inflammasome (Fig. 6C). In ad- marked Ca2+ influx and the magnitude of Ca2+ influx was small dition to NLRP3, we also investigated the role of SOCE in the compared with that induced by ionomycin as a control to mediate function of the NLRC4 inflammasome, which is activated by SOCE (Supplemental Fig. 4A, 4C). The Ca2+ signals following LPS bacterial flagellin and whose activation had been linked to Ca2+ 1210 STORE-OPERATED Ca2+ ENTRY AND MACROPHAGE AND DC FUNCTION Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 4. SOCE is not required for lysosomal trafficking of pathogens. (A–C) WT and Stim1fl/flStim2fl/fl Mx1-Cre BMDMs were incubated with 10 MOI of S. aureus-GFP for 2 h. Extracellular S. aureus was lysed with lysostaphin and lysosomes were stained with LysoTracker. (A) Representative differential interference contrast images of BMDMs, internalized S. aureus (green), and LysoTracker (red). Scale bars, 10 mm. Original magnification 360. (B) Colocalization of internalized S. aureus and LysoTracker was quantified after 60 and 120 min by blinded scoring; bar graphs represent mean 6 SEM of four independent experiments. (C) Number of intracellular (phagocytosed) S. aureus in individual BMDMs 15, 30, 60, and 120 min postinfection. Each dot rep- resents one cell. (D and E)WT,Stim1fl/fl Mx1-Cre,andStim1fl/flStim2fl/fl Mx1-Cre BMDMs were infected with 10 MOI of BCG-GFP for 3 h before extracellular bacteria were removed. Intracellular trafficking of BCG in BMDMs was measured 24 h later by GFP expression and staining of fixed cells with anti–LAMP-1 Ab. (D) Representative confocal microscopy images of internalized BCG-GFP (green) and LAMP-1 (red). Original magnification 360. (E) Quantification of data shown in (D) analyzing the MFI of LAMP-1 in the cell area surrounding BCG (as shown in the example in the upper panel; original magnification 360); the quantification represents .50 cells analyzed from two independent experiments; each dot represents a single intracellular BCG (lower panel). The Journal of Immunology 1211

FIGURE 5. Cytokine production by BMDMs and BMDCs is independent of SOCE. (A)WT and Stim1fl/flStim2fl/fl Vav-Cre BMDMs were stimulated with 1 mg/ml LPS, 50 mg/ml curdlan, or 5 MOI BCG for 16 h or left untreated (unstim.). Levels of TNF-a, IL-6, IL-10, IL-12p70, IL-2, and IL-12/23p40 in the cell supernatant were measured by ELISA. Bar graphs represent the Downloaded from mean 6 SEM of three mice per group. (B)WT and Stim1fl/flStim2fl/fl Vav-Cre BMDCs were stim- ulated with 1 mg/ml LPS, 100 nM CpG, 25 mg/ml zymosan, 50 mg/ml curdlan, or 1 mg/ml imiqui- mod for 16 h or left untreated (unstim.). Levels of TNF-a, IL-6, IL-10, IL-12p70, IL-2, and

IL-23p19 in cell supernatants were analyzed by http://www.jimmunol.org/ ELISA. Bar graphs represent the mean 6 SEM of eight mice per group. by guest on September 23, 2021

influx (52). Stimulation of LPS-primed BMDMs with the bacterial Vav-Cre and WT mice spontaneously showed a mature MHC toxin FlaTox that selectively activates the NLRC4 inflammasome class IIhiCD86hi phenotype without stimulation (Figs. 1G, 7A, 7B). (52) resulted in comparable IL-1b secretion in WT and STIM1/ Stimulation of immature BMDCs derived from WT and Stim1fl/flStim2fl/fl STIM2-deficient macrophages (Fig. 6F). Normal NLRP3 and NLRC4 Vav-Cre mice with different PRR ligands, including LPS, curdlan, inflammasome activation by LPS/ATP, LPS/MSU, and FlaTox, re- imiquimod, CpG, or zymosan, dramatically increased the percentage spectively, was also observed in BMDCs from Stim1fl/flStim2fl/fl of MHC class IIhiCD86hi mature DCs. However, no significant dif- Vav-Cre mice (Fig. 6G–I). Taken together, our findings show that ferences between WT and Stim1fl/flStim2fl/fl Vav-Cre BMDCs were Ca2+ influx and SOCE are not required for NLRP3 and NLRC4 observed (Fig. 7B). Next, we analyzed the effects of STIM1/STIM2 function. deletion in BMDCs on T cell activation. BMDCs from WT and Stim1fl/flStim2fl/fl Vav-Cre mice were incubated with OVA protein Ag presentation and stimulation of T cells by BMDCs is overnight, matured by stimulation with LPS, and cocultured with independent of STIM1 and STIM2 naive CFSE-labeled OT-II CD4+ T cells, which express a transgenic Many of the macrophage and DC functions we investigated above TCR specific for chicken OVA323–339.The proliferation of OT-II are required to initiate an adaptive immune response by T cells. T cells cocultured for 72 h with OVA-loaded Stim1fl/flStim2fl/fl Vav- This includes presentation of peptides from phagocytosed patho- Cre BMDCs was comparable to OT-II T cells incubated with WT gens on MHC class II complexes, expression of costimulatory BMDCs (Fig. 7C, 7D). molecules, and cytokine production. Ca2+ signals have been im- Because we had found that several BMDM and BMDC functions plicated in all of these steps (24–26, 36). Ca2+ influx via CRAC were impaired when cytosolic Ca2+ was chelated with BAPTA, we channels was suggested to regulate expression of MHC class II tested its effects on Ag presentation of DCs by treating WT and and the costimulatory molecules CD80 and CD86 on APCs (25, STIM1/STIM2-deficient BMDCs with BAPTA for 30 min before 71), and small interfering RNA–mediated suppression of STIM1 coculture with cognate T cells (Fig. 7C, 7D). BAPTA significantly and ORAI1 was shown to impair the maturation of human DCs (36). reduced T cell proliferation irrespective of whether T cells were We therefore investigated whether SOCE is required for the Ag- coincubated with WT or STIM1/STIM2-deficient BMDCs. These presenting function of DCs during an adaptive immune response. findings are consistent with the strongly impaired phagocytosis of A comparable small fraction of BMDCs from Stim1fl/flStim2fl/fl BAPTA-treated BMDCs (Supplemental Fig. 3D, 3E), which limits 1212 STORE-OPERATED Ca2+ ENTRY AND MACROPHAGE AND DC FUNCTION Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 6. NLRP3 and NLRC4 inflammasome activation does not require STIM1 and STIM2. (A) WT BMDMs were primed for 4 h with 10 mg/ml LPS with or without 2.2 mM EGTA, 10 mM BAPTA-AM, or 50 mM 2-APB followed by addition of 3 mM ATP to activate the NLRP3 inflammasome. After 30 min, cell supernatants were analyzed for IL-1b by ELISA. Data are normalized to IL-1b levels in LPS plus ATP–activated WT BMDMs without inhibition. Bar graphs represent the mean 6 SEM from six independent experiments. (B)Ca2+ signals in Fura-2–loaded BMDMs were measured after stimulation with 5 mM ATP in 2 mM Ca2+ Ringer solution using a FlexStation 3 plate reader. One representative experiment of two is shown. (C) BMDMs were primed for 4 h with 10 mg/ml LPS followed by stimulation with either 3 mM ATP for 30 min or 150 mg/ml MSU crystals for 4 h to activate the NLRP3 inflammasome. Secreted IL-1b in the supernatant was analyzed by ELISA. Bar graphs represent the mean 6 SEM of four experiments from two individual mice per group. n.d., not detectable. (D) BMDMs were left untreated or stimulated with 10 mg/ml LPS for 4 h followed by 3 mM ATP for 30 min to activate the NLRP3 inflammasome. Expression levels of procaspase-1 (∼45 kDa), cleaved caspase-1 (∼10 kDa), and actin (loading control) were analyzed by Western blotting. One representative experiment of three is shown. n.s., nonspecific. (E) BMDMs were primed with 10 mg/ml LPS for 15 min, 30 min, 1 h, 2 h, 4 h, and 16 h (o/n) alone or followed after 4 h by stimulation with 3 mM ATP for 15 and 30 min to activate the NLRP3 inflammasome. Intracellular pro–IL-1b (∼30 kDa) and actin levels were analyzed in whole-cell extracts by Western blotting. (F) BMDMs were primed for 4 h with 10 mg/ml LPS followed by stimulation with 3 mM ATP for 30 min to activate the NLRP3 inflammasome or 10 mg/ml LFn-FlaA together with 5 mg/ml PA (referred to as FlaTox) to activate the NLRC4 inflammasome. Secreted IL-1b in the supernatant was analyzed by ELISA. Bar graphs represent the mean 6 SEM from four individual mice per group. (G–I) BMDCs from WT and Stim1fl/flStim2fl/fl Vav-Cre mice were analyzed for Ca2+ influx (G) and activation of the NLRP3 (H and I) and NLRC4 (H) inflammasomes as described in (B), (C), and (F). Ca2+ traces are representative of two repeat experiments; bar graphs show the mean 6 SEM from five individual mice per group. Statistical significance was calculated with an unpaired Student t test. **p , 0.005, ***p , 0.001. the MHC class II–restricted presentation of cognate OVA- (Fig. 7E). In contrast, chelation of cytosolic Ca2+ in BMDCs derived peptides to OT-II T cells. In addition to proliferation, with BAPTA for 30 min before coculture with OT-II T cells we analyzed IFN-g production by OT-II T cells that were almost completely abolished IFN-g production. Taken to- coincubated with WT and STIM1/STIM2-deficient BMDCs for gether, our data demonstrate that the Ag-presenting function of 72 h and restimulated with PMA and ionomycin for 6 h but did DCs and their ability to stimulate T cells depend on cytosolic not observe a significant difference in the frequency of IFN-g+ Ca2+ signals but are independent of SOCE mediated by STIM1 T cells incubated with WT or STIM1/STIM2-deficient BMDCs and STIM2. The Journal of Immunology 1213 Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 7. Ag presentation by BMDCs and their ability to activate T cells is independent of STIM1 and STIM2. (A and B) Maturation of BMDCs does not require SOCE. WT and Stim1fl/flStim2fl/fl Vav-Cre BMDCs were left untreated (unstim.) or stimulated with 1 mg/ml LPS, 50 mg/ml curdlan, 1 mg/ml imiquimod, 100 nM CpG, or 25 mg/ml zymosan for 16 h and expression of MHC class II and CD86 on CD11c+ BMDCs was measured by flow cytometry. One representative experiment (A) and mean 6 SEM of averaged frequencies of MHC class IIhiCD86hi BMDCs from five individual experiments (B). (C–E) Ag presentation and T cell activation by BMDCs. WT and Stim1fl/flStim2fl/fl Vav-Cre BMDCs were maturated with 0.1 mg/ml LPS and incubated with 500 mg/ml OVA protein with or without 10 mM BAPTA-AM for 30 min. After washing, OVA-loaded BMDCs were cocultured at 5:1 and 1:1 ratios with CFSE- labeled WT OT-II CD4+ T cells for 3 d before CFSE dilution of CD4+ T cells was analyzed by flow cytometry. CFSE-labeled OT-II T cells were incubated with BMDCs without OVA protein (no OVA) or anti-CD3 and anti-CD28 mAbs (anti-CD3/28) as negative and positive controls, respectively. (C and D) Representative histogram plots (C) of proliferated CFSElo OT-II cells at different DC/T cell ratios and quantification (mean 6 SEM) of three experiments (D). (E) IFN-g production by OT-II cells was measured after coculture of BMDCs and CFSE-labeled OT-II cells at a 5:1 ratio for 72 h followed by restimulation with PMA plus ionomycin for 6 h in the presence of brefeldin A. IFN-g was measured by intracellular cytokine staining (left panel); the frequencies of IFN-g+ cells from three experiments are shown as mean 6 SEM (right panel). OT-II T cells incubated with BMDCs without OVA protein (no OVA) or stimulated with anti-CD3 and anti-CD28 mAbs (anti-CD3/28) in the presence of 20 ng/ml rIL-12 were used as negative and positive controls, respectively. Statistical significance was calculated with an unpaired Student t test. *p , 0.05, **p , 0.005, ***p , 0.001.

Discussion macrophages and DCs. Surprisingly, however, the complete lack of SOCE via CRAC channels is a conserved Ca2+ influx pathway in SOCE in these cells did not have a significant effect on any of their most, if not all, immune cells, but the contribution of SOCE to the innate immune functions, including FcR-dependent and -independent function of myeloid immune cells and innate immune responses is phagocytosis, phagolysosome fusion, cytokine production in response largely unknown. Patients lacking SOCE due to mutations in to various PRR ligands, activation of NLRP3 and NLRC4 inflam- ORAI1 or STIM1 genes are severely immunodeficient and suffer masomes,andAgpresentationtoTcells. In contrast, abolishing Ca2+ 2+ from recurrent, often life-threatening infections with viral, bac- signaling by chelation of [Ca ]i with BAPTA or the nonselective terial, mycobacterial, and fungal pathogens (6). Although many modulation of Ca2+ and other ion channels with 2-APB perturbed infections can be attributed to impaired T cell–mediated adaptive many macrophage and DC functions (Fig. 8). We therefore conclude immunity (6, 9, 10, 72), the patients’ frequent bacterial and my- that intracellular Ca2+ signals are essential for the function of mac- cobacterial (BCG) infections could be compounded by defective rophages and DCs, whereas SOCE mediated by STIM1/STIM2 is SOCE in innate immune cells resulting in an impaired function of dispensable. The apparently redundant role of SOCE in macrophages neutrophils, macrophages, and DCs. Consistent with the essential and DCs is in contrast to some earlier reports that had shown a role of role of STIM1 and STIM2 in SOCE in T cells (41) and B cells STIM1inphagocytosisbymacrophagesandinhumanDCmatura- (73), we found that conditional deletion of Stim1 and Stim2 genes tion and cytokine production (18, 36). We discuss below the potential in hematopoietic cells of mice completely abolishes SOCE in causes for these discrepancies. 1214 STORE-OPERATED Ca2+ ENTRY AND MACROPHAGE AND DC FUNCTION

Our data demonstrate that FcR-dependent and -independent in the liver (18). It is noteworthy that patients with loss-of- phagocytosis of latex beads, opsonized cells (RBCs), living bac- function mutations in the STIM1 gene that abolish SOCE de- teria (S. aureus), and mycobacteria (BCG) by murine macro- velop anti-RBC and anti-platelet autoantibodies and, in contrast to phages and DCs does not require STIM1 and STIM2 despite Stim12/2 mice, suffer from AIHA and thrombocytopenia. This abolished SOCE. Normal phagocytosis is not limited to in vitro– suggests that removal of autoantibody-bound RBCs and platelets cultured BMDMs because primary peritoneal macrophages by the mononuclear phagocyte system occurs normally in SOCE- isolated from STIM1/STIM2-deficient mice were able to phago- deficient patients (8, 72). cytose opsonized RBCs normally. Removal of extracellular Ca2+ The cause of the discrepant results obtained in Stim12/2 mice with EGTA partially impaired FcgR-dependent phagocytosis, and (18) compared with Stim1fl/flStim2fl/fl Vav-Cre or Stim1fl/flStim2fl/fl suppression of all intracellular Ca2+ signaling with BAPTA sig- Mx1-Cre mice used in this study and the STIM1 mutated human nificantly reduced phagocytosis. Our data are consistent with patients (8) is not understood. Different degrees of SOCE defi- observations that phagocytosis of IgG-coated beads and RBCs in ciency in cells from Stim12/2 (18) and Stim1fl/flStim2fl/fl Vav-Cre 2+ 2+ fl/fl fl/fl the absence of extracellular Ca or chelation of [Ca ]i was im- or Stim1 Stim2 Mx1-Cre mice are unlikely to account for the paired in mouse peritoneal macrophages and the macrophage cell divergent effects on phagocytosis because SOCE appears to be line J774 (15, 16). This dependence of FcgR-mediated phagocy- more profoundly impaired in STIM1/STIM2 double-deficient than tosis on intracellular Ca2+ was, however, not observed in other Stim12/2 macrophages. It is possible that peritoneal macrophages studies in which removal of extracellular Ca2+ or buffering in- are more dependent on SOCE than BMDMs; however, peritoneal tracellular Ca2+ had no effect on phagocytosis of IgG-coated macrophages from Stim1fl/fl Mx1-Cre and Stim1fl/flStim2fl/fl Mx1-Cre particles or RBCs (13, 14); the cause of these differences is not mice showed a normal ability to phagocytose Ab-opsonized RBCs. Downloaded from known. Importantly, our data demonstrate that phagocytosis is A potential explanation for the impaired phagocytosis by macro- independent of SOCE mediated by STIM1 and STIM2. This is in phages from Stim12/2 (18) but not Stim1fl/flStim2fl/fl Vav-Cre or contrast to a report showing strongly reduced phagocytosis of Stim1fl/flStim2fl/fl Mx1-Cre mice may be that Stim12/2 mice are on IgG2a and IgG2b opsonized RBCs in F4/80+ peritoneal macro- a different genetic background compared with the Stim1fl/flStim2fl/fl phages from Stim12/2 mice (18). STIM1-deficient macrophages Vav-Cre and Stim1fl/flStim2fl/fl Mx1-Cre mice, which are on a pure 2+ 2/2 had impaired Ca influx after crosslinking of FcgII/IIIa receptors. C57BL/6 background. It is possible that phagocytosis by Stim1 http://www.jimmunol.org/ In vivo, Stim12/2 mice injected with Abs against RBCs and plate- macrophages (18) is confounded by polymorphisms in modifier lets were protected from autoimmune hemolytic anemia (AIHA) genes that coregulate phagocytosis. A striking example of such and thrombocytopenia, respectively, which in the case of AIHA a modifier effect on phagocytosis was shown by Nuvolone et al. was attributed to impaired phagocytosis of RBCs by Kupffer cells (74) who demonstrated that the phagocytosis defect in Prnp2/2 mice reported in one study was not due to the lack of prion protein PrPc but was linked to a genetic polymorphism in the locus for signal regulatory protein a (Sirpa). A similar effect unrelated to STIM1 and SOCE might account for the phagocytosis defect in macrophages from Stim12/2 mice (18). Because the Stim12/2 by guest on September 23, 2021 mice used for these studies were bone marrow radiation chimeras, it is also possible that phagocytosis is affected by the irradiation- associated inflammation. One of the best characterized functions of SOCE in lymphocytes is the regulation of cytokine production through Ca2+-dependent transcription factors such as NFAT, ERK1, and NF-kB (64, 75). In T cells, the expression of IL-2, IL-4, IFN-g, TNF-a, IL-17, and many other cytokines is regulated by SOCE, and deletion of STIM1 or both STIM1 and STIM2 severely impairs cytokine production (41, 76, 77). We were therefore surprised to find nor- mal expression of IL-2, IL-6, IL-10, IL-12p70, IL-12/23p40, and TNF-a in STIM1/STIM2-deficient BMDMs and BMDCs fol- lowing stimulation with a variety of different PRR ligands that had been linked to Ca2+ signals (Fig. 8) (27–30, 32–34, 61, 63, 65). FIGURE 8. Intracellular Ca2+ signals but not SOCE regulate macro- Curdlan and zymosan are b glucans binding to the Dectin-1 re- phage and DC function. Engagement of FcgR by IgG and Dectin-1 by ceptor, which activates PLCg2 via the kinase Syk (34). The zymosan was shown to result in PLC-mediated production of IP3, which 2+ resulting IP3 production is thought to trigger a transient Ca re- binds to and facilitates opening of IP3R channels in the membrane of the ER. The resulting Ca2+ release from ER stores activates STIM1 and lease from ER stores and thus potentially SOCE (Fig. 8) (34). We 2+ STIM2 located in the ER membrane and causes the opening of CRAC observed Ca influx in WT BMDCs after zymosan but not channels (ORAI1, 2, and/or 3) in the plasma membrane, resulting in curdlan stimulation that appeared reduced in STIM1/STIM2- SOCE. Cells lacking STIM1/STIM2 have a selective defect in SOCE. deficient cells. Cytokine production in response to zymosan and Engagement of various PRRs such as TLR7 (by imiquimod), TLR9 (by curdlan, however, was not impaired. LPS binds to TLR4 and CD14 2+ CpG), and TLR4/CD14 (by LPS) was also reported to induce Ca signals, and was shown to induce Ca2+ signals in murine BMDCs (29, 30, although the channels that mediate these signals are unknown. Transient 35, 65). The LPS-induced expression of TNF-a and IL-6 was at- receptor potential (TRP) channels and P2X receptors contribute to Ca2+ tenuated by nonselective inhibition of Ca2+ channels with SKF- influx in macrophages and DCs but are not store operated. EGTA chelates 2+ extracellular Ca2+ and prevents SOCE via CRAC channels and Ca2+ influx 96365 (35). We found only a weak LPS-induced Ca response via other channels in the plasma membrane. 2-APB is a nonselective in a small fraction of WT BMDCs, which appeared absent in modulator of several ion channels in the plasma membrane and IP3Rs in STIM1/STIM2-deficient cells. However, we did not observe signif- the ER. BAPTA-AM chelates all cytosolic Ca2+ and thereby neutralizes the icant defects in LPS-induced cytokine production in STIM1/STIM2- effects of Ca2+ release from ER stores and extracellular Ca2+ influx. deficient DCs and macrophages. These findings are in contrast The Journal of Immunology 1215 to impaired expression of IL-10, IL-12, and IFN-g after LPS reduced T cell activation, suggesting that intracellular Ca2+ signals stimulation of human monocyte-derived DCs in the presence of but not SOCE are required for the APC function of DCs. 2-APB (36). 2-APB, however, is a nonselective modulator of several STIM1 and STIM2 mediate SOCE in macrophages and DCs but ion channels. It not only modulates CRAC channel function and their deletion and subsequent complete suppression of SOCE has SOCE (57, 78), but also that of TRPV channels (58, 79, 80), no effects on the function of these cells. At present, we cannot TRPM channels (81), TRPC channels (81), the potassium channel exclude that the SOCE-independent function of macrophages and

KCa3.1 that is expressed in macrophages (81, 82), GABAA DCs is specific to in vitro–differentiated BMDMs and BMDCs and receptors (83), and IP3Rs (reviewed in Ref. 84). 2-APB therefore that particular functions of primary macrophages and DCs in vivo has much broader effects on Ca2+ signaling than does the inhi- are dependent on SOCE. It is noteworthy, however, that peritoneal bition of SOCE alone. Although our data do not contradict a role macrophages isolated from STIM1/STIM2-deficient mice showed of Ca2+ signals in the regulation of cytokine expression in mac- no defect in the phagocytosis of opsonized RBCs. Additionally, + + rophages and DCs, they argue against an essential role of SOCE in CD11b and CD11c DCs isolated from MOG-immunized 2/2 2/2 cytokine production following PRR engagement. Stim1 bone marrow chimeric mice and Stim2 mice showed Extracellular Ca2+ as a danger signal was recently shown to be normal expression of costimulatory receptors MHC class II, CD80, required for the assembly and activation of the NLRP3 inflam- CD86, and CD40 and were able to activate MOG-specific T cells masome following binding to CASR on the cell surface (39, 40). in vitro (87), suggesting that STIM1 and STIM2 alone are not re- Ca2+ influx reportedly mediates activation of the NLRP3 inflam- quired for the Ag-presenting function of DCs. Future studies using masome because the nonselective ion channel inhibitor 2-APB mice with macrophage and DC-specific deletion of Stim1 and Stim2 blunted IL-1b secretion (38). This is consistent with our findings genes will have to address the role of SOCE in innate immune Downloaded from of a moderate reduction in NLRP3 activation when BMDMs were responses in vivo. 2+ stimulated in the presence of 2-APB and, more strikingly, abol- Our studies demonstrate that whereas intracellular Ca signals ished NLRP3 activation when intracellular Ca2+ was chelated with are required for several innate immune functions of macrophages BAPTA. Removal of extracellular Ca2+ with EGTA, however, had and DCs, SOCE mediated by STIM1 and STIM2 is dispensable almost no effect on IL-1b secretion, and STIM1/STIM2-deficient for these processes. This is in contrast to the important role of BMDMs showed normal caspase-1 cleavage and subsequent IL- SOCE in T cells and their ability to provide immunity to infection http://www.jimmunol.org/ 1b release. Because 2-APB nonselectively modulates many ion (9, 10) and to mediate inflammation in the context of autoim- channels besides CRAC channels, as discussed above, and BAPTA munity (47, 76, 87). Genetic or pharmacological inhibition of 2+ SOCE in lymphocytes prevents T cell–mediated autoimmunity suppresses all intracellular Ca signaling, these data collectively and inflammation in preclinical models of diseases such as multiple suggest that Ca2+ signals (potentially mediated by P2X7 receptors sclerosis (76, 87), inflammatory bowel disease (47), skin allograft or Ca2+ release from ER stores) but not SOCE are required for rejection (47), and allergic asthma (88), which may be used for the NLRP3 inflammasome activation. Given recent reports that K+ treatment of diseases in human patients. The beneficial effects of efflux but not Ca2+ influx is essential for NLRP3 activation (85, 2+ CRAC channel inhibition on proinflammatory T cells need to be

86), further studies are required on whether and how Ca sig- by guest on September 23, 2021 balanced, however, against their immunosuppressive effects that naling exactly contributes to NLRP3 inflammasome activation. result in increased susceptibility to infection. As CRAC channel Ca2+ signals have been implicated in the development and inhibitors are under development to treat autoimmune and inflam- function of DCs in response to microbial pathogens, and they may matory diseases, the dispensable role of SOCE in macrophages and thus play an important role in regulating innate immunity (31). DCs that we found in this study suggests that selective CRAC This view is supported by experiments in which enforcing Ca2+ channel inhibitors are unlikely to have significant side effects on signaling in DCs with Ca2+ ionophores promoted DC maturation innate immunity. whereas chelation of intracellular Ca2+ inhibited DC maturation (25, 36, 71). However, these experimental procedures are very Acknowledgments artificial and the Ca2+ signaling pathways that regulate DC de- We thank Dr. Russell Vance (University of California, Berkeley, CA) for velopment and function under more physiological conditions re- 2+ providing the FlaTox reagent and Dr. Victor Torres (New York University main unknown. Two studies have reported Ca currents in DCs School of Medicine) for GFP-expressing S. aureus. CD45.1+ congenic that resemble CRAC channel currents in T cells and observed that OT-II mice were a gift from Dr. Adrian Erlebacher (New York University stimulation of DC with IP3 or LPS induces SOCE (25, 35). Direct School of Medicine), and the CMG14-12 cell line was provided by Dr. Ken evidence for a role of SOCE in DCs comes from human Cadwell (New York University School of Medicine). We are grateful to monocyte-derived DCs in which small interfering RNA–mediated Dr. Masatsugu Oh-hora (Kyushu University, Kyushu, Japan) for generous fl/fl fl/fl fl/fl fl/fl knockdown of STIM1 or ORAI1 expression prevented LPS and help with Stim1 Stim2 Vav-Cre and Stim1 Stim2 Mx1-Cre mice TNF-a–induced maturation of DCs in vitro (36). Whether SOCE after hurricane Sandy decimated our mouse colony. is also required for other DC functions such as Ag presentation or inflammasome activation and especially for their function in vivo Disclosures remained unclear. We found that deletion of STIM1 and STIM2 S.F. is a cofounder of Calcimedica. The remaining authors have no financial completely abolishes SOCE in BMDCs. Surprisingly, however, conflicts of interest. the differentiation and maturation of DCs in response to a variety of TLR and other PRR ligands was unperturbed. Likewise, References STIM1/STIM2-deficient DCs incubated with OVA were able to 1. Feske, S., H. Wulff, and E. Y. Skolnik. 2015. Ion channels in innate and adaptive induce proliferation and IFN-g production of cognate T cells, immunity. Annu. Rev. Immunol. 33: 291–353. 2. Feske, S., Y. Gwack, M. Prakriya, S. Srikanth, S. H. Puppel, B. Tanasa, suggesting that none of the steps required for Ag presentation by P. G. Hogan, R. S. Lewis, M. Daly, and A. Rao. 2006. A mutation in Orai1 causes DCs (including Ag uptake, processing, and loading onto MHC immune deficiency by abrogating CRAC channel function. Nature 441: 179–185. class II as well as expression of costimulatory molecules and 3. Vig, M., C. Peinelt, A. Beck, D. L. Koomoa, D. Rabah, M. Koblan-Huberson, S. Kraft, H. Turner, A. Fleig, R. Penner, and J. P. Kinet. 2006. CRACM1 is cytokines) was significantly impaired in the absence of STIM1 and a plasma membrane protein essential for store-operated Ca2+ entry. Science 312: 2+ STIM2. In contrast, chelating [Ca ]i with BAPTA significantly 1220–1223. 1216 STORE-OPERATED Ca2+ ENTRY AND MACROPHAGE AND DC FUNCTION

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